Application of immune-inducing protein FvGH43A and its encoding gene in improving plant disease resistance

Abstract

This invention discloses the application of an immune-inducing protein FvGH43A and its encoding gene in improving plant disease resistance, belonging to the field of plant immune-inducing technology. This invention discovered a novel protein FvGH43A with plant immune-activating function from *Fusarium oxysporum*, which can induce programmed cell death in plants, promote reactive oxygen species bursts, and promote the expression of defense genes in plants, thereby effectively activating plant immune activity and exhibiting broad-spectrum resistance. The protein FvGH43A of this invention significantly improves the resistance level of plants to *Fusarium* and *Phytophthora*, indicating its development potential and application value as a broad-spectrum immune-activating agent for crops.

Description

Technical Field

[0001] This invention relates to the field of plant immune induction technology, specifically to the application of an immune induction protein FvGH43A and its encoding gene in improving plant disease resistance. Background Technology

[0002] Fusarium pseudoverticum ( Fusarium verticillioides As a highly pathogenic fungus, it has become a significant biological stressor restricting global agricultural production. This pathogen has a wide host range, infecting many important economic crops such as corn, wheat, rice, cotton, tomatoes, and peppers. Its damage to corn is particularly prominent, causing various diseases including seedling blight, ear rot, stem rot, and seed rot. Furthermore, the fungus secretes various toxins when infecting plants, posing a serious and persistent threat to human and animal health.

[0003] Currently, the control of Fusarium wilt in agricultural production mainly relies on the breeding of resistant varieties and the application of chemical pesticides. However, crop varieties resistant to this fungus are extremely scarce, with a severe lack of highly resistant varieties. On the other hand, the long-term and extensive use of chemical pesticides has led to a series of serious problems, including excessive pesticide residues in agricultural products, rapid increase in pathogen resistance, continuous pollution of soil and water environments, and rising control costs year by year.

[0004] In nature, the immune response is crucial for plant resistance to diseases. Studies have shown that proteins derived from certain pathogens can act as activation signals for plant immunity, inducing systemic and broad-spectrum disease resistance, thereby significantly enhancing plant resistance to pathogen infection and damage. This discovery provides a key material basis for the development of novel plant immune inducers. Compared to traditional chemical pesticides, immune-activating proteins offer advantages such as high efficiency, environmental friendliness, safety, and broad spectrum. The development and utilization of immune proteins hold promise for opening new pathways for the green control of crop diseases. However, the inducing proteins that rapidly and effectively activate plant immunity have not yet been fully explored, greatly limiting the research and development of bio-based immune inducers and severely hindering the innovative application of protein-based plant immune inducers and their derivatives in the green control of crop diseases. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of the immune-inducing protein FvGH43A in improving plant disease resistance. This invention discovers a novel protein with plant immune-activating function from *Fusarium oxysporum*, which can provide a core target and material basis for developing efficient and safe green control products and application technologies for crop diseases, and has significant practical application value.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the application of the immune-inducing protein FvGH43A in improving plant disease resistance; said immune-inducing protein FvGH43A is a protein as shown in (A1) or (A2) below:

[0008] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing;

[0009] (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0010] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the above applications, the immune-inducing protein FvGH43A enhances plant disease resistance by activating plant immunity.

[0012] Furthermore, the immune-inducing protein FvGH43A activates plant immunity through at least one of the following pathways (1)-(3):

[0013] (1) It causes programmed cell death in plants;

[0014] (2) Triggers a surge of reactive oxygen species in plants;

[0015] (3) Promote the expression of defense genes in plants.

[0016] Preferably, the plant's defense genes include: ZmLOX3, ZmPAL1, ZmPOD, ZmPR-1, ZmLOX4 and ZmLOX5 .

[0017] Preferably, the plant is corn, tobacco, cotton, tomato, chili pepper, peanut, eggplant, sweet potato or soybean.

[0018] Preferably, the disease resistance is against diseases caused by Fusarium and / or Phytophthora; for example: corn stalk rot, wheat stem base rot, tobacco Fusarium root rot, solanaceous plant wilt, tobacco black shank, etc.

[0019] A second aspect of the invention provides FvGH43A The application of the coding gene in the following (1) or (2):

[0020] (1) Improve plant disease resistance;

[0021] (2) Cultivating disease-resistant plant varieties;

[0022] FvGH43A The coding gene is a nucleic acid molecule as shown in i) or ii) below:

[0023] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2;

[0024] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.1.

[0025] In the above applications, by promoting FvGH43A Encoding gene expression to enhance plant disease resistance.

[0026] Preferred, promoting FvGH43A The substance encoding gene expression is any one of the following:

[0027] C1) contains FvGH43A Expression cassettes encoding genes;

[0028] C2) contains FvGH43A Recombinant vectors encoding genes, or recombinant vectors containing the expression cassette described in C1);

[0029] C3) contains FvGH43A Recombinant microorganisms encoding genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

[0030] Preferably, the plant is corn, tobacco, cotton, tomato, chili pepper, peanut, eggplant, sweet potato or soybean.

[0031] Preferably, the disease resistance is against diseases caused by Fusarium and / or Phytophthora.

[0032] The beneficial effects of this invention are:

[0033] (1) The present invention discovered a new protein FvGH43A with plant immune activation function from Fusarium tumefaciens. It can induce programmed cell death in plants, promote the burst of reactive oxygen species in plants and promote the expression of defense genes in plants, thereby effectively activating plant immune activity and having broad-spectrum resistance.

[0034] (2) The protein FvGH43A of the present invention significantly improves the resistance level of plants to Fusarium and Phytophthora, indicating that it has the development potential and application value as a broad-spectrum immune activating agent for crops. Attached Figure Description

[0035] Figure 1 Electrophoresis image of FvGH43A protein.

[0036] Figure 2Inoculation with FvGH43A induced programmed cell death in tobacco cells.

[0037] Figure 3 Inoculation with FvGH43A induced programmed cell death in various plants; In the figure, A: corn leaf; B: cotton leaf; C: tomato leaf; D: pepper leaf; E: peanut leaf; F: eggplant leaf; G: sweet potato leaf; H: soybean leaf; To the left of the dashed line in the leaf is the blank control group inoculated with 20 μL of 10 μM PBS buffer (pH=7.4); To the right of the dashed line is the treatment group inoculated with 20 μL of 10 μM FvGH43A.

[0038] Figure 4 Inoculation with FvGH43A induced reactive oxygen species (ROS) bursts in tobacco and maize. In the figure, A represents the ROS burst in tobacco detected by DAB plant tissue staining, and B represents the ROS burst in maize detected by luminol chemiluminescence.

[0039] Figure 5 Inoculation with FvGH43A induced upregulation of maize defense genes; in the figure, AF represent... ZmLOX4, ZmLOX5, ZmPR-1, ZmLOX3, ZmPOD and ZmPAL1 The relative expression levels; the data include the mean ± standard error of three replicates (** for p ≤ 0.01; *** for p ≤ 0.001; **** for p ≤ 0.0001).

[0040] Figure 6 Example 3: Maize phenotypic diagrams of processed A, B, C, and D.

[0041] Figure 7 Statistical results of maize disease incidence and disease index in processing A, B, C, and D in Example 3; the data include the mean ± standard error of the three replicate results (*** for p ≤ 0.001; **** for p ≤ 0.0001).

[0042] Figure 8 Tobacco phenotypes of A, B, C, and D processed in Example 4.

[0043] Figure 9 Example 4: Statistical results of tobacco incidence and disease index of A, B, C, and D; the data include the mean ± standard error of the three replicate results (*** for p ≤ 0.001; **** for p ≤ 0.0001). Detailed Implementation

[0044] 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.

[0045] As mentioned earlier, immune-activating proteins have the advantages of high efficiency, environmental friendliness, safety, and broad spectrum. The development and utilization of immune proteins are expected to open up new pathways for the green control of crop diseases. However, the inducing proteins that effectively activate plant immunity have not been fully explored and identified, which greatly limits the research and development process of bio-derived immune inducers and seriously hinders the innovative application of protein-based plant immune inducers in the green control of crop diseases.

[0046] Therefore, this invention conducts an in-depth study of *Fusarium verticillatum*. FVEG_10625 is a hypothetical protein of unknown function in *Fusarium verticillatum*. This invention performs structural analysis on the FVEG_10625 protein, truncates it, and names the truncated protein FvGH43A. Its amino acid sequence is shown in SEQ ID NO.1, as follows:

[0047] LALSKRDSPVLPGLWADPNIAIVDKTYYIFPTTDGFEGWGGNVFYWWKSKDLVSWTKSDKPFLTLNGTNGNVPWATGNAWAPAFAARGGKYYFYHSGNNPSVSDGHKSIGAAVADHPEGPWKAQDKPMIKGTSDEEIVSNQAIDPAAFEDPETGKW YIYWGNGVPIVAELNDDMVSLKAGWHKITGLQNFREGLFVNYRDGTYHLTYSIDDTGSENYRVGYATADNPIGPWTYRGVLLEKDESKGILATGHNSIINIPGTDEWYIAYHRFHIPDGNGYNRETTIDRVPIDKDTGLFGKVTPTLQSVDPRPL.

[0048] To investigate the function of the FvGH43A protein, this invention amplified the gene encoding the FvGH43A protein. FvGH43A Its nucleotide sequence is shown in SEQ ID NO.2; specifically as follows:

[0049] ctcg ccctcagcaa acgggatagc cctgtcctcc ccggcctctg ggcggaccccaacatcgccatcgtcgacaa gacatactac atcttcccta ccaccgacgg tttcgaaggctggggcggca acgtcttctactggtggaaa tcaaaagatc tcgtatcatg gacaaagagcgacaagccat tccttactct caatggtacgaatggcaacg ttccctgggc tacaggtaatgcctgggctc ctgctttcgc tgctcgcgga ggcaagtattacttctacca tagtgggaataatccctctg tgagtgatgg gcataagagt attggtgcgg cggtggctgatcatcctgaggggccgtgga aggcacagga taagccgatg atcaagggaa cttctgatgaggagattgtcagcaaccagg ctatcgatcc cgctgccttt gaagaccctg agactggaaagtggtatatctactggggaa acggtgtccc cattgtcgca gagctcaacg acgacatggtctctctcaaagcaggctggc acaaaatcac aggtcttcag aatttccgcg agggtcttttcgtcaactatcgcgatggaa catatcatct gacatactct atcgacgata cgggctcagagaactatcgcgttgggtacg ctacggcgga taaccccatt ggaccttgga catatcgtggtgttcttctggagaaggacg aatcgaaggg cattcttgct acgggacata actccatcatcaacattcctggaacggatg agtggtatat cgcgtatcat cgcttccata ttcccgatggaaatgggtataatagggaga ctacgattga tagggtaccc atcgacaagg atacgggtttgtttggaaaggttacgccga ctttgcagag tgttgatcct aggcctttgtag.

[0050] The FvGH43A protein was expressed and purified. The effect of the purified FvGH43A protein on plant immune activation was then investigated, and the results showed that FvGH43A protein could significantly activate the plant's immune response. This invention further investigated the effects of FvGH43A protein on maize diseases caused by *Fusarium verticillatum* and tobacco diseases caused by *Phytophthora infestans*, and the results showed that FvGH43A protein could significantly improve the disease resistance of maize and tobacco, thus leading to this invention.

[0051] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels. Wherein:

[0052] The *Fusarium pseudocortex* used in the embodiments of this invention ( F. verticillioides The standard strain of *Fusarium verticillatum* is Fv7600. *Phytophthora tobaccoii* ( Phytophthora nicotianae (Provided by the National Plant Protection Microbial Germplasm Resource Bank (Shandong))

[0053] The immune-inducing protein FvGH43A used in this invention is a truncated FVEG_10625 protein, the amino acid sequence of which is shown in SEQ ID NO.1. It can be obtained through exogenous expression in eukaryotes or prokaryotes, or through existing chemical synthesis techniques. In the embodiments of this invention, the FvGH43A protein was obtained through exogenous expression in yeast.

[0054] The culture medium and its components used in the embodiments of this invention are as follows:

[0055] MD medium: 13.4 g / L yeast basic nitrogen source (purchased from Sigma-Aldrich, USA, catalog number Y1250); 0.4 mg / L biotin; 20 g / L glucose.

[0056] YPD medium: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar powder; all are mass / volume ratios, in g / 100 mL.

[0057] BMGY medium: yeast extract: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 7.4 phosphate buffer, glycerol: 1.0 mL, add distilled water to 100 mL.

[0058] BMMY medium: yeast extract: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 7.4 phosphate buffer, after high-temperature sterilization, add 1 mL of methanol to every 100 mL of medium.

[0059] Example 1: Expression and purification of the immune-inducing protein FvGH43A from Fusarium verticillatum.

[0060] 1. Construction of yeast expression vector carrying target gene:

[0061] 20 μL of *E. coli* culture containing the pPIC9K plasmid was incubated in LB broth containing kanamycin (final concentration 50 μg / mL) at 37°C with shaking for 12 h. Plasmids were extracted according to the instructions of the TIANGEN plasmid mini-prep kit. Restriction endonucleases were selected. Eco RI (Thermo Scientific) and Not I (Thermo Scientific) performed double digestion of the plasmid to expose the ligation ends of the fragment. If the reaction was complete, the remaining reaction solution was electrophoresed and the digested products were recovered by gel electrophoresis.

[0062] Construct a complete expression vector according to the Ligation-Free Cloning System kit (New England Biolabs) instructions, and attach the expression vector to both ends. Eco RI and Not The enzyme amplification product from the I restriction site was ligated into the double-digested yeast expression vector pPIC9K.

[0063] FvGH43A The amplification product encoding the gene was obtained as follows: RNA was extracted from the hyphae of the standard strain Fv7600 of *Fusarium verticillatum*, and a design was performed. FvGH43A Gene-specific primers (shown in SEQ ID NO.3 and SEQ ID NO.4) were used to amplify the gene using RT-PCR, resulting in primers with primers at both ends bearing [specific primers]. Eco RI and Not The amplification product of the I restriction site is ligated into the double-digested yeast expression vector pPIC9K to obtain the yeast expression plasmid pPIC9K / carrying the target gene. FvGH43A .

[0064] FvGH43A The sequences of the specific primers encoding the gene are as follows:

[0065] FvGH43A -F:CCGGAATTCCTCGCCCTCAGCAAACG; (SEQ ID NO.3)

[0066] FvGH43A -R: TTGCGGCCGCCTAGTGGTGGTGGTGGTGGTGCAAAGGCCTAGGATCAACAC. (SEQ IDNO.4)

[0067] The amplification reaction system is shown in Table 1.

[0068] Table 1 PCR reaction system

[0069]

[0070] Yeast expression plasmid pPIC9K / FvGH43A Transplanted with E. coli E. coli After T1 competent cells were cultured at 28°C for 12 h, single colonies were picked and cultured in 1 mL of LB liquid medium containing 50 μg / mL kanamycin at 37°C with shaking for 6 h. 1.5 μL of the bacterial culture was used as a template for PCR verification and the product was sent for sequencing. If the sequence result was correct, the yeast expression vector carrying the target gene was obtained.

[0071] 2. Construction of engineered yeast strains:

[0072] The yeast expression vector carrying the target gene was linearized and dephosphorylated, and then transformed into competent yeast cells to obtain engineered yeast strains; the details are as follows:

[0073] (1) Linearization and dephosphorylation of expression vectors

[0074] Take 20 μL of the yeast expression vector (pPIC9K / ) containing the target gene prepared in Example 1. FvGH43A Escherichia coli culture was incubated in 20 mL of LB broth containing 50 μg / mL kanamycin at 37°C with shaking for 12 h. Plasmids were extracted according to the instructions of the TIANGEN plasmid mini-prep kit and their concentration was determined. Linearized restriction endonucleases were selected. Pme I (Thermo Scientific) and dephosphorylase AP (Thermo Scientific) were used to perform the reaction according to the instructions of the restriction endonuclease kit.

[0075] (2) Electrocution

[0076] Before electroporation, a usable GS115 strain and fresh competent yeast cells must be obtained. Aliquot 80 μL of the prepared competent yeast cells into each tube, add the linearized expression vector plasmid, mix gently, and incubate on ice for 5 min. Transfer the mixture to a pre-chilled electroporation cuvette, electroporate at 300 V for 15 ms, immediately add 1 mol / L sorbitol solution (on ice), mix well, and incubate at 28°C for 60 min. Spread 300 μL onto MD medium and incubate at 28°C for 2–3 days.

[0077] (3) Screening and validation of engineered yeast strains

[0078] Single colonies were selected and screened on YPD medium containing 150 μg / mL and 180 μg / mL kanamycin. Colonies that grew rapidly on both concentration plates were selected, and PCR verification was performed using 5'AOX1 and 3'AOX1 primers. The products were then sequenced. If the fragment size and sequencing results were correct, the bacterium could be used as a fermentation engineer.

[0079] The 5'AOX1 primer sequence is: GACTGGTTCCAATTGACAAGC; (SEQ ID NO.5)

[0080] 3'AOX1 primer sequence: GCAAATGGCATTCTGACATCC. (SEQ ID NO.6)

[0081] 3. Protein expression and purification:

[0082] (1) Fermentation of engineered yeast

[0083] The screened and identified engineered yeast strains were streaked onto YPD medium and cultured at 28°C for 2 days. Afterward, the activated engineered yeast strains were picked and inoculated into BMGY medium. The culture was incubated at 28°C with shaking at 200 rpm for 22–24 h. After centrifugation at 4200 rpm, the bacterial culture was transferred to BMMY medium to begin induction of expression. 1 mL of methanol was added every 24 h for 7 consecutive days. The supernatant crude enzyme solution was collected at 10°C for 8000 rpm for 20 min.

[0084] (2) Ammonium sulfate precipitation and dialysis of crude enzyme solution

[0085] Add dried ammonium sulfate powder to the crude enzyme solution until saturated, then let it stand overnight at 4°C. Centrifuge at 10°C / 8000 rpm / 20 min, discard the supernatant, add 10 mL of PBS buffer (pH=7.4), and after all the precipitated protein has dissolved, transfer it to a dialysis bag and dialyze in PBS buffer (pH=7.4) for 24 h. Centrifuge at 4°C / 8000 rpm / 15 min to collect the supernatant.

[0086] (3) Protein purification

[0087] The purification process for the preparation of recombinant proteins with histidine tags (His) employed a His Trap. TM The HP (Cytiva) AKTA protein purification system was used. The column was washed with deionized water and equilibrated with nickel using 10 μM PBSA buffer (pH=7.4). In column chromatography experiments, the dialyzed enzyme solution was injected into the column, and the flow rate should be kept as low as possible to ensure successful operation. After loading, HisTrap was used. TM The HP chromatography column was equilibrated with PBSB buffer (pH=7.4) at a flow rate maintained below 1.5 mL / min. After equilibration, elution was performed using PBSC buffer (pH=7.4). The flow solution was then transferred to a dialysis bag and dialyzed six times (15 min each time) with 10 μM PBS buffer (pH=7.4).

[0088] (4) SDS-PAGE validation analysis

[0089] The molecular weight of FvGH43A protein was determined by SDS-PAGE denaturing electrophoresis. The procedure and staining / destaining process followed the instructions of the gel preparation kit (Solarbio). After electrophoresis, the stacking gel was removed, and the separating gel was stained and destained. After destaining, the gel was placed on a film viewing lamp for observation and analysis to determine if the desired enzyme was expressed and whether the separation and purification were successful.

[0090] The results are as follows Figure 1 As shown, there is only one independent protein band, which is clear, bright, and of the expected size, confirming the correct expression of the protein.

[0091] The purified protein was subjected to amino acid sequencing, and its amino acid sequence is shown in SEQ ID NO.1. The purified protein was named immune-inducing protein FvGH43A.

[0092] Example 2: Activation of plant immunity by the immune-inducing protein FvGH43A

[0093] 1. The immune-inducing protein FvGH43A induces programmed cell death in tobacco and maize leaf cells.

[0094] Programmed cell death (PCD) is one of the core mechanisms of plant immune response and a hallmark phenomenon of immune response.

[0095] 20 μL of 1–500 μM FvGH43A protein was inoculated into 1-month-old tobacco leaves, with tobacco leaves inoculated with 20 μL of 10 μM PBS buffer (pH=7.4) serving as a control. Forty-eight h after inoculation, programmed cell death at the inoculation site was detected using trypan blue staining.

[0096] The results are as follows Figure 2 As shown, when the concentration of FvGH43A protein is 1 μM, it can induce programmed cell death in tobacco cells. As the protein concentration increases, the color of the inoculation site gradually deepens, indicating that a low concentration (1 μM) of FvGH43A protein can activate the plant immune response, and the immune activation effect increases with the increase of protein concentration. Furthermore, a low concentration (1 μM) can effectively activate the plant immune response.

[0097] 2. The immune-inducing protein FvGH43A induces programmed cell death in various plants.

[0098] To further verify the immune activation effect of FvGH43A protein on different host plants, healthy corn, cotton, tomato, pepper, peanut, eggplant, sweet potato, and soybean were selected as experimental subjects. 20 μL of 10 μM FvGH43A protein was inoculated on the right side of each plant leaf, while 20 μL of 10 μM PBS buffer (pH=7.4) was inoculated on the left side of the same leaf as a control. Cell death at the inoculation sites was observed 48 h after inoculation.

[0099] The results are as follows Figure 3 As shown, FvGH43A protein can induce programmed cell death in various plants, indicating that FvGH43A protein has a broad spectrum of ability to activate plant immunity.

[0100] 3. The immune-inducing protein FvGH43A triggers reactive oxygen species bursts in tobacco and corn.

[0101] To further verify the activation of plant immunity by FvGH43A, healthy one-month-old tobacco plants were selected as experimental subjects. 20 μL of 10 μM FvGH43A protein was inoculated on the right side of a tobacco leaf, and 20 μL of 10 μM PBS buffer (pH 7.4) was inoculated on the left side of the same leaf. Three hours after inoculation, the leaves were immersed in DAB staining solution and stained at room temperature in the dark for 4-12 hours, until the positive areas turned dark brown, and the remaining areas were pale yellow, nearly colorless, or the plant's natural color. The tobacco leaves were removed with tweezers, rinsed 3-5 times in distilled water, blotted dry on filter paper, and then immersed in tissue destaining solution in a water bath at 70-80℃ for 20-40 minutes until the tissue background color was completely removed. After cooling, the tobacco leaves were removed, rinsed 3-5 times in distilled water, blotted dry on filter paper, and then transferred to an appropriate amount of tissue preservation solution for 10-30 minutes before being photographed.

[0102] The results are as follows Figure 4 As shown in Figure A, tobacco leaves inoculated with FvGH43A protein showed a distinct dark brown color, while tobacco leaves inoculated with PBS buffer (pH=7.4) did not show a distinct brown color, indicating that FvGH43A can induce a burst of reactive oxygen species in plants.

[0103] One-week-old healthy maize leaves were collected, and leaf discs were punched using a 4 mm punch and rinsed in 9 cm petri dishes. These discs were then transferred to 96-well plates containing 200 μL of ultrapure water per well and soaked overnight. The reaction solution was prepared in advance: 100 μL of 50 μM luminol reagent (Bio-Rad), 1 μL of 50 μg / mL horseradish peroxidase (HRP, Sigma), and 10 μL of 10 μM FvGH43A protein. Fluorescence signals were then detected using a Glomax20 / 20 luminescence detector (Promega). 10 μL of 10 μM PBS buffer (pH = 7.4) was used as a control to replace the FvGH43A reaction solution. Three biological replicates were performed for each sample.

[0104] The results are as follows Figure 4 As shown in Figure B, inoculation with FvGH43A can rapidly trigger a surge of reactive oxygen species (ROS) in maize leaves, while the ROS in the control group showed no significant change. This indicates that inoculation with FvGH43A induces early immune signal transduction in maize, thereby rapidly triggering the production of ROS.

[0105] 4. The immune-inducing protein FvGH43A promotes the expression of maize resistance genes.

[0106] 20 μL of 1 μM FvGH43A protein was inoculated into leaves of healthy one-week-old maize. Total RNA was extracted from leaves at 0 h and 3 h after treatment. The expression of multiple maize defense response genes was detected using RT-qPCR. Maize defense genes ZmLOX3 , ZmPAL1 , ZmPOD , ZmPR-1 , ZmLOX4 , ZmLOX5 The primer designs are shown in Table 2. ZmEF1a This is an internal reference gene. The RT-qPCR reaction system and amplification conditions are shown in Tables 3 and 4, respectively.

[0107] Table 2 List of primer designs for defense genes

[0108]

[0109] Table 3 RT-qPCR reaction system

[0110]

[0111] Table 4 RT-qPCR amplification reaction conditions

[0112]

[0113] The results are as follows Figure 5 As shown, inoculation with the immune-inducing protein FvGH43A can significantly enhance maize defense genes. ZmLOX3 , ZmPAL1 , ZmPOD , ZmPR-1 , ZmLOX4 , ZmLOX5 The level of expression.

[0114] Example 3: Effects of the immune-inducing protein FvGH43A on maize disease resistance

[0115] 1. Test method:

[0116] Using 2-week-old maize leaves with similar growth as the experimental subjects, the experiment was designed with the following treatments:

[0117] Treatment A: Use only 50 mL of clean water to irrigate the roots as a control;

[0118] Treatment B: Use 50 mL of Fusarium oxysporum Fv7600 spore suspension (spore concentration 1×10⁻⁶). 6 (cfu / mL) root irrigation;

[0119] Treatment C: Inoculate maize leaves with 20 μL of 1 μM FvGH43A protein. After 24 h, inoculate with 50 mL of Fusarium verticillatum Fv7600 spore suspension (spore concentration 1×10⁻⁶).6 (cfu / mL) root irrigation;

[0120] Treatment D: Inoculate maize leaves with 20 μL of 1 μM FvGH43A protein, and irrigate the roots with 50 mL of water 24 h later.

[0121] Each treatment consisted of 30 corn plants, and the disease incidence was observed after 48 hours. Disease classification was performed according to existing techniques (“Research on Identification and Integrated Control Technology of Maize Stalk Rot Pathogen” [J]. Seed Science and Technology, 2025, 43(16):134-136.).

[0122] Grade 0 indicates no disease; Grade 1 indicates slight discoloration at the base of the stem; Grade 2 indicates lesions covering less than 1 / 4 of the stem base; Grade 3 indicates lesions covering 1 / 4 to 1 / 2 of the stem base; Grade 4 indicates lesions covering 1 / 2 to 3 / 4 of the stem base; Grade 5 indicates lesions covering more than 3 / 4 of the stem base or the plant has collapsed.

[0123] Calculate the incidence rate and disease index using the following formula:

[0124] Incidence rate (%) = (Number of infected plants ÷ Total number of plants) × 100%

[0125] Disease index = ∑(Number of diseased plants at each level × Corresponding level) ÷ (Total number of plants surveyed × Highest level) × 100

[0126] 2. Test Results:

[0127] Phenotypic photos of maize in each treatment group after 48 h are shown below. Figure 6 As shown, the statistical results of incidence and disease index are as follows: Figure 7 As shown in the figure, the results showed that maize treated with FvGH43A had significantly enhanced disease resistance compared to the control, proving that FvGH43A significantly increased maize's resistance to Fusarium oxysporum.

[0128] Example 4: Effect of immune-inducing protein FvGH43A on tobacco disease resistance

[0129] 1. Test method:

[0130] Using one-month-old tobacco leaves with similar growth as the test subjects, the experiment was conducted with the following treatments:

[0131] Treatment A: Root irrigation with only 50 mL of clean water served as a control;

[0132] Treatment B: Use 50 mL of Phytophthora spore suspension (spore concentration of 1×10⁻⁶). 6 (cfu / mL) root irrigation;

[0133] Treatment C: Tobacco leaves were inoculated with 20 μL of 1 μM FvGH43A protein. After 24 h, the leaves were treated with 50 mL of Phytophthora spore suspension (spore concentration of 1×10⁻⁶). 6 (cfu / mL) root irrigation;

[0134] Treatment D: Inoculate tobacco leaves with 20 μL of 1 μM FvGH43A protein, and irrigate the roots with 50 mL of water 24 h later.

[0135] Each treatment consisted of 30 tobacco plants, and disease incidence was observed after 48 hours. Disease severity was classified according to GB / T 23222-2008 "Classification and Investigation Methods for Tobacco Diseases and Pests".

[0136] Grade 1: Stem lesions do not exceed one-third of the stem circumference, or the leaves wither below one-third of the stem circumference.

[0137] Grade 3: Stem lesions surround one-third to one-half of the stem circumference, or one-third to one-half of the leaves are slightly wilted, or a few leaves on the lower part of the stem show lesions.

[0138] Grade 5: Stem lesions extend beyond half the stem circumference but do not completely encircle the stem circumference, or half to two-thirds of the leaves wither.

[0139] Level 7: Stem lesions completely surround the stem circumference, or more than two-thirds of the leaves wither.

[0140] Level 9: The diseased plants are basically dead.

[0141] Calculate the incidence rate and disease index using the following formula:

[0142] Incidence rate (%) = (Number of infected plants ÷ Total number of plants) × 100%

[0143] Disease index = ∑(Number of diseased plants at each level × Corresponding level) ÷ (Total number of plants surveyed × Highest level) × 100

[0144] 2. Test Results:

[0145] Phenotypic photos of tobacco from each treatment group after 48 h are shown below. Figure 8 As shown, the statistical results of incidence and disease index are as follows: Figure 9 As shown, compared with the control, tobacco treated with FvGH43A showed significantly enhanced disease resistance, proving that FvGH43A significantly improved the resistance level of tobacco to Phytophthora.

[0146] 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 the immune-inducing protein FvGH43A in improving plant disease resistance, characterized in that, The immune-inducing protein FvGH43A is the protein shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); The plant is corn or tobacco; the disease resistance is resistance to diseases caused by Fusarium verticillatum or Phytophthora tobaccoii.

2. The application according to claim 1, characterized in that, The immune-inducing protein FvGH43A enhances plant disease resistance by activating plant immunity.

3. The application according to claim 2, characterized in that, The immune-inducing protein FvGH43A activates plant immunity through at least one of the following pathways (1)-(3): (1) It causes programmed cell death in plants; (2) Triggers a surge of reactive oxygen species in plants; (3) Promote the expression of defense genes in plants.

4. FvGH43A The application of the coding gene in the following (1) or (2): (1) Improve plant disease resistance; (2) Cultivating disease-resistant plant varieties; FvGH43A The coding gene is a nucleic acid molecule as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 1; The plant is corn or tobacco; the disease resistance is resistance to diseases caused by Fusarium verticillatum or Phytophthora tobaccoii.

5. The application according to claim 4, characterized in that, By promoting FvGH43A Encoding gene expression to enhance plant disease resistance.

6. The application according to claim 5, characterized in that, Promote FvGH43A The substance encoding gene expression is any one of the following: C1) contains FvGH43A Expression cassettes encoding genes; C2) contains FvGH43A Recombinant vectors encoding genes, or recombinant vectors containing the expression cassette described in C1); C3) contains FvGH43A Recombinant microorganisms encoding genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2).

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