Wheat dwarf bush smut effect protein gene g1217 and application thereof

Abstract

The present application relates to the field of plant pathology and genetic engineering technology, and specifically provides a wheat dwarf smut effector protein gene g1217 and application thereof. The nucleotide sequence of the effector protein gene g1217 is shown as SEQ ID NO:1, and the encoded protein sequence is shown as SEQ ID NO:2. The present application verifies that the effector protein can effectively inhibit BAX-induced programmed cell death of plants through transient expression technology, indicating that it plays an important role in inhibiting plant defense response. In addition, the subcellular localization results show that the protein is located on the cell membrane. The present application provides an important gene resource and theoretical basis for in-depth study on the pathogenic mechanism of wheat dwarf smut, revealing the pathogen-plant interaction mechanism, and cultivating wheat varieties resistant to wheat dwarf smut.

Description

Technical Field

[0001] This invention belongs to the fields of plant pathology and genetic engineering technology, and relates to a wheat dwarf smut effector protein gene g1217 and its application. Background Technology

[0002] Ustilago tritici ( Tilletia controversa Wheat dwarf smut induced by *Trismus kuhn* (TCK) is a quarantine disease in my country and an important international quarantine disease. This disease is devastating to wheat production, reducing yield by more than 80% and even leading to total crop failure. Infected wheat grains also contain trimethylamine, producing a foul, rotten, fishy odor, severely affecting flour quality, and potentially causing poisoning in humans and animals. Currently, most production methods rely on biochemical pesticides to control wheat dwarf smut, lacking safe and effective control measures. This pathogen already poses a risk of introduction, colonization, and spread in my country; therefore, elucidating the pathogenic mechanism of *Trismus kuhn* at the genetic level is of great significance for the effective prevention and control of wheat dwarf smut.

[0003] During infection, pathogens secrete small proteins called "effect proteins," which target key immune pathways in host cells, suppressing the plant's defense response and thus promoting infection and colonization. Therefore, identifying and characterizing the effector protein genes of *Ustilago maydis* (wheat dwarf smut) is crucial for elucidating its pathogenic mechanism and developing novel disease control strategies.

[0004] When expressed in plants, BAX proteins (pro-apoptotic members of the Bcl-2 family) induce programmed cell death, a process similar to the hypersensitive necrosis response triggered by pathogen infection. Transient co-expression of BAX proteins in tobacco leaves using the BAX system has become a classic experimental system for screening and verifying the inhibitory effector proteins of pathogens on plant immune function. Existing technologies include reports on cloning effector protein genes from *Ustilago maydis*. For example, Chinese patent CN115896132B discloses a *Ustilago maydis* effector protein gene g10960. This study verified through a tobacco transient expression system that the effector protein encoded by the g10960 gene can effectively inhibit BAX-induced programmed cell death in plants, indicating its important role in suppressing plant defense responses and providing gene resources for the study of the pathogenic mechanism of *Ustilago maydis*.

[0005] However, the pathogenic process of the pathogen is the result of the synergistic action of multiple effector proteins, and different effector proteins may target different host immune pathways or different nodes in the same pathway. Therefore, research on a single effector protein alone is insufficient to fully reveal the complex infection mechanism of *Ustilago maydis*. Discovering more effector protein genes with inhibitory plant immune functions will not only help to deepen the understanding of the interaction network between the pathogen and the host plant, but also provide more gene resources and targets for the subsequent development of broad-spectrum and durable disease-resistant wheat varieties. Summary of the Invention

[0006] The purpose of this invention is to provide a novel effector protein gene, g1217, from *Ustilago maydis* and its applications. Through in-depth analysis of the *Ustilago maydis* transcriptome data, a new effector protein gene, named g1217, was discovered. Transient expression in tobacco confirmed that this effector protein effectively inhibits BAX protein (a pro-apoptotic member of the Bcl-2 family)-induced programmed cell death (PCD) in plants, indicating its important role in suppressing plant defense responses.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention first provides a wheat dwarf smut effector protein gene g1217, the nucleotide sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0008] The present invention further provides the application of the overexpression of the wheat dwarf smut effector protein gene g1217 in inhibiting BAX protein-induced necrosis of tobacco leaf tissue, wherein the gene g1217 inhibits BAX protein-induced programmed cell death in tobacco plants.

[0009] This invention also provides the application of biomaterials overexpressing the *Ustilago maydis* effector protein gene g1217 in inhibiting BAX protein-induced necrosis of tobacco leaf tissues, wherein the biomaterials inhibit BAX protein-induced programmed cell death in tobacco plants; the biomaterials are any one of A1) to A3) below: A1) An expression cassette containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1; A2) A recombinant vector containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1; A3) A recombinant microorganism containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1, or a recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2), wherein the microorganism is Agrobacterium.

[0010] The present invention also provides a method for inhibiting tobacco leaf tissue necrosis caused by BAX protein, wherein the method involves overexpressing the wheat smut effector protein gene g1217 in tobacco, and the gene g1217 inhibits programmed cell death in tobacco plants induced by BAX protein.

[0011] Preferably, the overexpression of the wheat dwarf smut effector protein gene g1217 in tobacco involves transferring a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO:1 into Agrobacterium, introducing it into tobacco, and increasing the expression level of the g1217 gene in tobacco.

[0012] As a preferred embodiment of the present invention, the recombinant expression vector is constructed through the following steps: Step 1: Using cDNA synthesized by reverse transcription of total RNA extracted from wheat dwarf smut fungus as a template, PCR amplification was performed using primers g1217F1 and g1217R1 to obtain the g1217 gene fragment; the nucleotide sequence of primer g1217F1 is shown in SEQ ID NO:3, and the nucleotide sequence of primer g1217R1 is shown in SEQ ID NO:4; Step 2: Use restriction endonucleases Cla I and Sal I The pGR107 vector was double-digested, and the linearized pGR107 vector fragment was recovered. Step 3: Ligate the g1217 gene fragment from Step 1 with the linearized pGR107 vector fragment from Step 2, transform it into competent E. coli cells, and obtain positive clones through screening and sequencing verification, thus obtaining the recombinant expression vector pGR107-g1217.

[0013] More preferably, in step one, the PCR reaction system is as follows: 2× Master Mix 25 μL, 10 μM upstream primer g1217F1 2 μL, 10 μM downstream primer g1217R1 2 μL, cDNA template 0.5 μg, ddH2O added to 50 μL; the PCR reaction program is as follows: 95℃ for 5 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0014] In this embodiment, the g1217 gene was introduced into an expression vector and transformed into Agrobacterium GV3101. Then, the bacterial culture of Agrobacterium GV3101 containing the g1217 gene expression vector was injected into tobacco leaves in good growth condition after 4-6 weeks. Through transient expression in tobacco leaves, the effector protein of BAX protein was used to induce apoptosis, which verified that the effector protein expressed by the g1217 gene can effectively inhibit BAX-induced programmed cell death in plants, indicating that the effector protein plays an important role in inhibiting plant defense response.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a novel wheat dwarf smut effector protein gene g1217, the nucleotide sequence of which is shown in SEQ ID NO:1, and the protein sequence it encodes is shown in SEQ ID NO:2. The discovery of this gene enriches the gene resources of wheat dwarf smut effector proteins and provides a new molecular tool for in-depth research on the pathogenic mechanism of the pathogen.

[0016] (2) The present invention verified the function of the g1217 gene using a transient expression system in tobacco. The experimental results showed that the effector protein expressed by the g1217 gene could effectively inhibit BAX protein-induced programmed cell death in plants. BAX-induced cell death is similar to the mechanism of hypersensitive necrosis response in plants caused by pathogen infection. Therefore, the g1217 gene plays an important role in inhibiting plant defense response.

[0017] (3) This invention provides the application of the g1217 gene in inhibiting tissue necrosis in tobacco leaves, and provides corresponding biological materials and methods, providing new candidate genes and technical pathways for the subsequent development of wheat varieties resistant to wheat dwarf smut. Compared with the g10960 gene disclosed in the prior art, the g1217 gene is a new effector protein gene with a completely different sequence. The synergistic effect or functional difference between the two needs further research, but the discovery of this invention undoubtedly provides an important supplement to the comprehensive analysis of the pathogenic mechanism of wheat dwarf smut.

[0018] (4) The present invention also provides specific methods for constructing recombinant expression vectors and methods for transient expression of tobacco mediated by Agrobacterium, which are simple to operate and have good reproducibility, providing clear technical guidance for those skilled in the art to implement the present invention. Attached Figure Description

[0019] Figure 1 The results are obtained by agarose gel electrophoresis of the PCR amplification product of the g1217 gene of wheat dwarf smut.

[0020] Figure 2This is a phenotypic observation of the transient expression of the *Ustilago maydis* g1217 gene in tobacco leaves; A: Schematic diagram of tobacco leaf injection; gene represents *Agrobacterium* bacterial suspension containing the g1217 gene, eGFP represents *Agrobacterium* bacterial suspension containing the eGFP gene, Buffer represents tobacco injection solution (blank control), gene+BAX represents an equal volume mixture of *Agrobacterium* bacterial suspension containing the g1217 gene and *Agrobacterium* bacterial suspension containing the BAX gene, eGFP+BAX represents an equal volume mixture of *Agrobacterium* bacterial suspension containing the eGFP gene and *Agrobacterium* bacterial suspension containing the BAX gene, Buffer+BAX represents an equal volume mixture of Buffer and *Agrobacterium* bacterial suspension containing the BAX gene; B: Symptom diagram of tobacco leaves after injection; C: Symptom diagram of tobacco leaves after trypan blue staining and decolorization after injection.

[0021] Figure 3 These are the subcellular localization results of the g1217 effector protein from wheat smut. Detailed Implementation

[0022] The present invention will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0023] Biomaterials and Carriers Ustilago tritici ( Tilletia controversa Kuhn, TCK): The wheat smut strain used in the following examples is from the literature Xu TS, Qin DD, Muhae Ud Din G, Liu TG, Chen W, Gao L. Characterization of histological changes at the tillering stage (Z21) inresistant and susceptible wheat plants infected by Tilletia controversa Kühn. BMC Plant Biol, 2021, 18; 21(1): 49. This strain is available to the public from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The applicant promises to release the biological material to the public within twenty years from the date of application.

[0024] Escherichia coli Trans2-Blue competent cells: purchased from Beijing TransGen Biotech Co., Ltd.

[0025] Agrobacterium GV3101 (pJIC SA-Rep) competent cells: purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0026] The tobacco used in the following examples is Benedictine tobacco (Benjamin Butyl) Nicotiana benthamiana (Provided by this laboratory).

[0027] Potato X virus vector pGR107: Purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd. The potato X virus vector pGR107 is a binary expression vector containing the CaMV 35S promoter and kanamycin resistance gene. The GenBank accession number for the vector sequence is AY297842.1, and its full length is 438 bp.

[0028] PBin-GFP vector: a commercially available vector.

[0029] Main reagents and solutions Universal DNA purification and recovery kits and plasmid mini-extraction rapid kits were purchased from Beijing Tiangen Biotech Co., Ltd. 2× Pro Taq Master Mix (dye plus) was purchased from Hunan Aikerui Biotechnology Co., Ltd. High-fidelity enzyme 2× Phanta ® Flash Master Mix (Dye Plus), ClonExpress ® The Ultra One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. The penicillin-streptolysin bispecific antibody was purchased from Gibco, catalog number 15140-122.

[0030] (1) 0.25% NaClO: 250μL NaClO to 100mL.

[0031] (2) 50× TAE Buffer: Dissolve 242g Tris and 37.2g EDTA-Na2·2H2O in 600mL of deionized water. Add 57.1mL of glacial acetic acid (anhydrous acetic acid) to the dissolved liquid and bring the volume up to 1L. Adjust the pH to 8.5 with NaOH.

[0032] (3) 2% soil extract culture medium: The soil used must first be sterilized in an autoclave at 60℃~100℃ for 30~60min. Take 75g of soil and filter it through 8 layers of gauze with 500mL of boiled distilled water, make up to 1000mL, and sterilize at 120℃ for 20min. (20g of agar needs to be added to the solid culture medium) (4) LB medium: Weigh 3g sodium chloride, 1.5g yeast extract, and 3g tryptone, bring the volume to 300mL, and sterilize at 120℃ for 20min. (For solid medium, add 4.5g agar powder) (5) Kanamycin antibiotic (50mg / ml): Weigh 2.5g of kanamycin and place it in a 50mL centrifuge tube. First, add 40mL of sterile distilled water, mix and dissolve thoroughly, and then bring the volume to 50mL. Filter with a 0.22μm filter membrane to sterilize, aliquot and store at -20℃.

[0033] (6) Rifampicin antibiotic (20mg / mL): Weigh 0.2g of rifampicin into a 50mL centrifuge tube, add 10mL of DMSO (dimethyl sulfoxide), filter to sterilize, and then dispense into aliquots. Store at -20℃.

[0034] (7) Tobacco injection (Buffer): Take 5 mL of 1 M MgCl2 stock solution, 5 mL of 1 M MES stock solution, and 0.5 mL of 0.2 M AS (acetylsyl syringone) stock solution, add sterile water to make up to 500 mL, mix well and set aside.

[0035] (8) Trypan blue staining solution: 10 mL lactic acid, 10 mL glycerol, 10 mL phenol, 10 mg trypan blue, 10 mL deionized water. First, completely dissolve the trypan blue in deionized water, then add it to the other solutions and mix well. Store at 4°C.

[0036] (9) Trypan blue decolorizing solution: 250g of hydrated trichloroacetaldehyde, diluted to 100mL with deionized water, shaken at low speed at 37℃ or at 65℃ until completely dissolved.

[0037] PCR primers Table 1 Primers required for vector construction

[0038] Main instruments

[0039] PCR instrument, manufactured by Thermo Fisher Scientific, USA. Centrifuge and pipettes, manufactured by eppendrof Life Sciences, Germany. 50ml centrifuge, manufactured by Sigma, Germany. Electrophoresis apparatus, manufactured by Bio-Rad, USA. Autoclave, manufactured by Zealway, USA. GHB-202 thermostatic metal bath, manufactured by Hangzhou Borui Technology Co., Ltd. Thermostatic water bath, manufactured by Shanghai ZJ Biotech Co., Ltd. 85-2 digital display thermostatic magnetic stirrer, manufactured by Ronghua Instrument Manufacturing Co., Ltd., Jintan City, Jiangsu Province. Large-capacity full-temperature shaker, manufactured by Suzhou Peiying Experimental Equipment Co., Ltd. Clean bench, manufactured by Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Confocal laser microscope (Zeiss 980).

[0040] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, 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 invention pertains.

[0041] Explanation of the sequence list

[0042] SEQ ID NO:1 Open reading frame sequence (360bp) of the g1217 gene. ATGCGATTCACCATCCTCGCCACTGCGCTCGCTGCCACCGCCACCCTCGTCAGCGCCGTGCCCGTCCCCAAGGGCGACGCCATCCACCTCCAGCTCCCCTTCGGCTACAACACCTTTTGACGTCGCCATCCCGGACTGGTTCCAGTCCAACGACGTCTGCCCCGACCTCAAGGTCTCCTGT GTCAAGGATGGCAAGACCGTCGGTGACATCGGCAAGAAGCCTTACCAGTGGGAGGGCATCAAGAAGCTGTGTAAGCAGTCCGAGCCCGAGAAGAACACAGCCGAGTGCAACCTCCGCTTTGCCGACAAGTGCGCCGCTCACTGCGAAGCCCAAGGACCCCTTGGCGGCCACGGCATCTAA The protein sequence encoded by the g1217 gene (119aa) is shown in SEQ ID NO:2. MRFTILATALAATATLVSAVPAPKGDAIHLQLPFGYNTFDVAIPDWFQSNDVCPDLKVSCVKDGKTVGDIGKKPYQWEGIKKLCKQSEPEKNTAECNLRFADKCAAHCEAQGPLGGHGI SEQ ID NO:3 Primer g1217F1 (42 bp) TCAGCACCAGCTAGCATCGATATGGTGCCCGCTCCCAAGGGC SEQ ID NO:4 Primer g1217R1 (40bp) AACCGTTCATCGGCGGTCGACTTAGATGCCGTGGCCGCCA SEQ ID NO:5 Primer pGR107F (22 bp) ATAGCAGTCATTAGCACTTCCT SEQ ID NO:6 Primer pGR107R (22bp) GTTAGCAGGTGGACTGTTGTTA SEQ ID NO:7 Primer g1217F2 (42 bp) ATTTACGAACGATAGGGTACCATGGTGCCCGCTCCCAAGGGC SEQ ID NO:8 Primer g1217R2 (40bp) GCCCTTGCTCACCATGGATCCGATGCCGTGGCCGCCAAGG SEQ ID NO:9 Primer pBin107F (20bp) ACAATCCCACTATCCTTCGC SEQ ID NO:10 Primer pBin107R (19 bp) AAGTCGTGCTGCTTCATGT Example 1. Cloning and functional verification of the g1217 gene of wheat dwarf smut. 1. Cultivation of wheat smut

[0043] (1) Take 5-6 wheat dwarf smut galls and put them into a 2mL centrifuge tube. Add 2mL of sterile distilled water and let stand at room temperature for 30min. (2) Use tweezers to peel off the seed coat and pick out the small grains. Filter the impurities through 150-mesh gauze and then centrifuge. (3) Centrifuge at 6000 rpm for 1 min, discard the supernatant, add 2 mL of 0.25% NaClO for 10 min to sterilize; (4) Centrifuge at 6000 rpm for 1 min, discard the supernatant, and add sterile distilled water; (5) Repeat steps (3) and (4) above twice; (6) Using an optical microscope and a hemocytometer, the concentration of wheat smut teliospores was adjusted to the order of magnitude of 1×10⁻⁶ after dilution treatment. 6 cells / mL; (7) Add Gibco (1% by volume) to the 2% soil extract culture medium, pour the plates, and spread 220 μL of teliospore suspension in each petri dish. Place the plates in an artificial incubator at 5℃, 1Lx and 50% relative humidity under full light for about 45 days. Then observe the germination status of teliospores under an optical microscope. Collect the teliospores after they have grown to the stage of infecting mycelia for the extraction of RNA from wheat smut.

[0044] 2. Extraction of RNA and synthesis of cDNA from *Ustilago maydis* (wheat smut).

[0045] RNA was extracted from the mycelial hyphae of *Ustilago maydis* using the Trizol method. The specific steps are as follows: (1) Collect the germinating mycelium of wheat dwarf smut, place it in an RNase-free 2mL spiral tube, and quickly place it in liquid nitrogen. Use a high-throughput rapid sample preparation instrument to grind the sample into powder. (2) Add 1 mL (the amount can be adjusted according to the sample amount) of TRIzol reagent (Invitrogen) to the well-ground mycelial powder, shake gently to mix well and let it stand at room temperature for 5-10 min; (3) Add chloroform to the liquid that has been lysed with TRIzol reagent at a ratio of 0.2 mL chloroform per 1 mL TRIzol reagent, shake vigorously for 15 s, extract, let stand at room temperature for 3 min, and centrifuge at 12000 rpm for 15 min at 4℃. (4) After centrifugation, the supernatant was transferred to a new 1.5 mL RNase-Free centrifuge tube, and an equal volume of isopropanol was added for sedimentation. After mixing, the mixture was placed on ice for 10 min, and then centrifuged at 12000 rpm for 15 min at 4 °C. (5) Discard the supernatant, add 1 mL of 75% ethanol (prepared with anhydrous ethanol and RNase-free water), mix by inversion, centrifuge at 4°C and 12000 rpm for 5 min to remove residual liquid as much as possible. (6) Repeat the previous step, and then dry in the clean bench for 5 to 10 minutes until the precipitate becomes transparent; (7) Add an appropriate amount of RNase-free water to dissolve the RNA, store it in a -80℃ refrigerator for later use.

[0046] The total RNA obtained was used to synthesize first-strand cDNA using the TaKaRa RNA Reverse Transcription Kit (catalog number: RR047A) according to the kit instructions, and the cDNA samples were used as gene cloning templates. The resulting cDNA samples were stored at -20°C.

[0047] 3. Cloning of the target gene

[0048] Based on transcriptome data from *Ustilago maydis*, we discovered an effector protein gene, named g1217. The nucleotide sequence of the open reading frame (ORF) of the g1217 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0049] The N-terminal signal peptide sequence of the effector protein encoded by the g1217 gene was predicted using the online analysis software SignalP 5.0. The gene sequence encoding the signal peptide was then removed, and primers g1217F1 and g1217R1 (primer sequences are shown in Table 1) were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. Using reverse-transcribed cDNA of *Ustilago maydis* as a template, the target gene fragment was amplified by PCR using the synthesized primers. The PCR reaction system is shown in Table 2.

[0050] Table 2 PCR reaction system for the target gene

[0051] The PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 2 min, 35 cycles; 72℃ for 10 min. The PCR products were detected by 1.0% agarose gel electrophoresis (150V, 30 min), and the results are as follows. Figure 1 As shown, the product size is correct. The target band was excised from the gel and recovered using a universal DNA purification and recovery kit (Tiangen Biotech, catalog number: DP214-03) according to the kit instructions. The recovered product was stored at -20°C.

[0052] 4. Enzyme digestion of the vector

[0053] Take 10 µL of *E. coli* culture containing the pGR107 vector and place it in 10 mL of LB broth containing kanamycin (50 mg / mL). Incubate at 37°C with a shaker at 200 rpm for 8–10 h. Then, extract plasmids according to the instructions using a rapid plasmid miniprep kit (Tiangen Biotech, catalog number: DP105-03). Based on the vector and gene sequence, use... Cla I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0144) and Sal The pGR107 plasmid was double-digested using enzyme I (Thermo Fisher Scientific, Inc., Catalog No.: FD0644). The digestion reaction system is shown in Table 3.

[0054] Table 3 Cla I and Sal I. Double enzyme digestion reaction system

[0055] The prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour. After the reaction, the digestion products were detected by 1.0% agarose gel electrophoresis. The target band was cut off with a sterile blade and recovered using a universal DNA purification and recovery kit (Tiangen Biotech, DP214-03), following the kit instructions. The recovered product was stored at -20°C.

[0056] 5. Ligation of the target gene and the vector

[0057] (1) Using Vazyme's ClonExpress ® The Ultra One Step Cloning Kit is a seamless ligation kit that ligates purified linear vectors with target gene fragments.

[0058] (2) The recombinant connection system is shown in the table below: Table 4. Ligation reaction system of plasmid pGR107 and target gene

[0059] (3) After gently mixing the prepared system, place it in a 50°C metal bath for 10 minutes, and then place it on ice for 2 minutes.

[0060] (4) Immediately transfer 5-10 µL of recombinant plasmid into 100 µL of Escherichia coli Trans2-Blue competent cells in a molten state and incubate on ice for 30 min.

[0061] (5) Heat shock in a water bath at 42°C for 45 seconds, then place on ice for 2-3 minutes.

[0062] (6) Add 800µL of antibiotic-free liquid LB medium to the clean bench.

[0063] (7) Shake the bacteria at 37℃ and 200rpm for 1 hour. Use a sterile glass rod to spread the bacterial solution evenly on an LB solid plate containing kanamycin (50mg / ml). Incubate in an inverted position in a 37℃ incubator overnight.

[0064] (8) Select 6-8 single clones for colony PCR verification. The PCR primers are pGR107F and pGR107R (primer sequences are shown in Table 1). The PCR reaction system is as follows: Table 5. Bacterial PCR Reaction System

[0065] The PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 2 min, 35 cycles; 72℃ for 10 min. The amplified products were sequenced and verified by Beijing Qingke Biotechnology Co., Ltd.

[0066] 6. Transformation of Agrobacterium competent cells

[0067] The positive colonies that were correctly sequenced in the previous step were subjected to plasmid extraction using a rapid plasmid miniprep kit (Tiangen Biotech, catalog number: DP105-03). The extracted recombinant plasmid pGR107-g1217 was then introduced into Agrobacterium GV3101 (pJIC SA-Rep) competent cells. The Agrobacterium transformation method is as follows: (1) Take out the competent Agrobacterium cells stored at -80℃, let them thaw at room temperature or in your palm for a period of time, and insert them into ice when they are in a state of ice-water mixture.

[0068] (2) Then add the extracted plasmid to the competent cells. Add 0.01 to 1 µg of plasmid to every 100 µL of competent cells. After adding, gently tap the bottom of the tube to mix.

[0069] (3) Place it on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 37°C for 5 minutes, and ice bath for 5 minutes in sequence.

[0070] (4) Add 700µL of LB liquid medium without resistance to the competent cells and culture them in a shaker at 200rpm at 28℃ for 2-3 hours; (5) Centrifuge the shaken bacterial solution at 6000 rpm for 1 min to collect the bacterial cells. Resuspend the bacterial cells in about 100 µL of supernatant and spread the bacterial solution on an LB solid plate containing rifampicin (25 mg / ml) and kanamycin (50 mg / ml) using a sterile glass rod.

[0071] (6) Invert the plate and incubate it in a 28°C incubator for 2-3 days. When a single colony grows on the plate, pick 8 single colonies for bacterial PCR verification. The PCR reaction system and reaction procedure are the same as above.

[0072] (7) Pick the PCR-verified colonies into 10 mL of liquid LB medium containing rifampicin (25 mg / ml) and kanamycin (50 mg / ml), and incubate at 28°C and 200 rpm for 2 days. Store the bacterial solution for later use.

[0073] 7. Transient gene expression in tobacco

[0074] The BAX gene (Gengbank accession number: L22472.1) is a member of the Bcl-2 family of apoptosis genes. The protein expressed by this gene induces apoptosis, similar to the hypersensitive necrosis response mechanism in pathogen-plant interactions. The eGFP gene (Gengbank accession number: EU746493.1) is an enhanced green fluorescent protein gene that fluoresces under blue light excitation. Agrobacterium GV3101 containing the BAX gene expression vector (PGR107-BAX) and Agrobacterium GV3101 containing the eGFP gene expression vector (PGR107-eGFP) were strains prepared and preserved in our laboratory. The PGR107-BAX vector was constructed using… Sal I and Cla The PGR107 vector and the BAX gene fragment were double-digested with restriction endonucleases, respectively. Then, the BAX gene fragment was inserted into the PGR107 vector via a ligation reaction. The construction method of the PGR107-eGFP vector is as follows: using... Sal I and Cla The PGR107 vector and the eGFP gene fragment were double-digested with restriction endonucleases, and then the eGFP gene fragment was inserted into the PGR107 vector through a ligation reaction.

[0075] The method for transient gene expression in tobacco is as follows: (1) Agrobacterium containing the g1217 gene expression vector, Agrobacterium containing the eGFP gene expression vector, and Agrobacterium containing the BAX gene expression vector were inoculated into 10 mL of LB liquid medium containing rifampicin (25 mg / mL) and kanamycin (50 mg / mL), respectively, and cultured by shaking at 28 °C and 200 rpm for 2 days.

[0076] (2) Collect bacterial cells by centrifugation at 5000 rpm for 10 min and resuspend the cells in Buffer (Tobacco Injection) (10 mmol / L MES, 200 μmol / L AS, 10 mmol / L MgCl2).

[0077] (3) Repeat the above steps, wash three times, and then activate in a dark environment at 28°C for 3 hours. Adjust the volume to OD with an appropriate amount of tobacco injection solution. 600 =0.3~0.5.

[0078] (4) Using a 1 mL sterile syringe with the needle removed, draw an appropriate amount of the bacterial suspension. Infiltrate *Agrobacterium* onto tobacco leaves that are 4–6 weeks old and in good growth condition. Inject the bacterial suspension onto the underside of the tobacco leaves using the infiltration method. Use the 6-zone injection method, following the... Figure 2As shown in Figure A, the injection method involved injecting Agrobacterium-containing bacterial solution, Agrobacterium-containing eGFP bacterial solution, and Buffer (tobacco injection solution) into the left side of the tobacco leaf from top to bottom, and marking the injection areas. Each tobacco leaf had six injection areas, with the Buffer in the lower left corner serving as a blank control. Three to five leaves were injected per tobacco plant. Each sample was repeated three times.

[0079] (5) 24 hours after vaccination, follow the instructions. Figure 2 As shown in Figure A, injection methods were performed on the right side of the tobacco leaf (to the right of the area marked in the previous step), from top to bottom: gene+BAX (a mixture of equal volumes of Agrobacterium-containing solutions of the g1217 gene and Agrobacterium-containing solutions of the BAX gene), eGFP+BAX (a mixture of equal volumes of Agrobacterium-containing solutions of the eGFP gene and Agrobacterium-containing solutions of the BAX gene), and Buffer+BAX (a mixture of equal volumes of buffer and Agrobacterium-containing solutions of the BAX gene). Here, gene represents the g1217 gene. The tobacco leaves were then incubated at 25°C for 3–8 days. The phenotype of necrosis in the injected areas was observed, data were recorded, and photographs were taken.

[0080] (6) Mix trypan blue staining solution with anhydrous ethanol at a volume ratio of 1:1. Place the tobacco leaves showing symptoms into the mixed staining solution, boil them in boiling water for 5 minutes, stain them overnight, and then place the leaves into a decolorizing solution for decolorization. Take a picture after decolorization is completed.

[0081] The results are as follows Figure 2 B (symptom diagram of tobacco leaves after injection) and Figure 2 Figure C (symptoms of tobacco leaves after trypan blue staining and decolorization) shows the following: In the left injection area of ​​the tobacco leaf: no cell necrosis was observed at the sites where Buffer was injected alone (blank control), eGFP was injected alone, or the g1217 gene was injected alone. In the right injection area: the Buffer+BAX injection site and the eGFP+BAX injection site both showed symptoms of leaf tissue drying and necrosis. However, after injection of Agrobacterium tumefaciens gene + BAX, no tissue necrosis was observed in the corresponding tobacco parts. These phenotypic symptoms indicate that BAX can successfully induce an allergic necrosis response in tobacco cells; the absence of cell necrosis at the site where the g1217 gene was injected alone indicates that the effector protein expressed by the g1217 gene cannot induce an allergic necrosis response in tobacco; and the absence of tissue necrosis at the site where g1217+BAX was injected indicates that the g1217 gene can effectively inhibit BAX-induced programmed cell death (PCD). Therefore, the effector protein expressed by the g1217 gene plays an important role in inhibiting plant defense responses.

[0082] Example 2. Subcellular localization of the effector protein of *Ustilago maydis* g1217. 1. Cloning of the target gene Using wheat smut cDNA as a template, PCR amplification was performed using primers g1217F2 and g1217R2 (primer sequences are shown in Table 1). The PCR reaction system and procedure were the same as in Example 1 (cloning of the target gene).

[0083] 2. Enzyme digestion of the vector First, the *E. coli* bacterial culture containing the pBin-GFP vector was amplified. Then, plasmid extraction was performed using a rapid plasmid mini-prep kit (Tiangen Biotech, catalog number: DP105-03) according to the kit instructions. Based on the vector and gene sequence, plasmids were extracted using... Kpn I enzyme (Thermo Fisher Scientific, USA, catalog number: FD0524) and BamH The pBin-GFP plasmid was double-digested using enzyme I (Thermo Fisher Scientific, Inc., catalog number: FD0054). The digestion system is as follows: Table 6 Kpn I and BamH I. Double enzyme digestion reaction system

[0084] The prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour. After the reaction, the enzyme digestion products were detected by 1.0% agarose gel electrophoresis. The target band was cut off with a sterile blade and recovered using a universal DNA purification and recovery kit (Tiangen Biotech, catalog number: DP214-03).

[0085] 3. Ligation of the target gene and the vector (1) Using Vazyme's ClonExpress ® The Ultra One Step Cloning Kit is a seamless ligation kit that ligates purified linear vectors with target gene fragments.

[0086] (2) The recombinant connection system is as follows: Table 7 Recombinant Linkage System

[0087] (3) After gently mixing the prepared system, place it in a 50°C metal bath for 10 minutes, and then place it on ice for 2 minutes.

[0088] (4) Immediately transfer 5-10 µL of recombinant plasmid into 100 µL of Escherichia coli Trans2-Blue competent cells in a molten state and incubate on ice for 30 min.

[0089] (5) Heat shock in a water bath at 42°C for 45 seconds, then place on ice for 2-3 minutes.

[0090] (6) Add 800µL of antibiotic-free liquid LB medium to the clean bench.

[0091] (7) Shake the bacteria at 37℃ and 200rpm for 1 hour. Use a sterile glass rod to spread the bacterial solution evenly on an LB solid plate containing kanamycin (50mg / ml). Incubate in an inverted position in a 37℃ incubator overnight.

[0092] (8) Select 6-8 single clones for bacterial culture PCR verification. The PCR primers are pBin107F and pBin107R (primer sequences are shown in Table 1). The PCR reaction system is shown in Table 8.

[0093] Table 8 Recombinant Connection System

[0094] The PCR reaction program was as follows: 95℃ for 5 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 2 min, 35 cycles; 72℃ for 10 min. The amplified products were sequenced and verified by Beijing Qingke Biotechnology Co., Ltd.

[0095] 4. Transformation of Agrobacterium competent cells The positive colonies that were correctly sequenced in the previous step were subjected to plasmid extraction using a rapid plasmid miniprep kit (Tiangen Biotech, catalog number: DP105-03) according to the kit instructions. The extracted recombinant plasmid pBin-g1217 was then transformed into Agrobacterium GV3101 (pJIC SA-Rep) competent cells. The Agrobacterium transformation method was the same as in Example 1 (Transformation of Agrobacterium competent cells).

[0096] 5. Transient gene expression in tobacco Successfully transformed Agrobacterium colonies were placed in 10 mL of LB broth containing rifampin and kanamycin (Kan: 50 μg / mL; Rif: 25 μg / mL) for shake culture and propagation. The culture was then incubated at 28°C with shaking until OD... 600 When the concentration of bacteria is 1.0–2.0, the cells are collected by centrifugation at 5000 rpm for 5 min. The cells are resuspended in an equal volume of tobacco injection buffer (10 mmol / L MES, 200 μmol / L AS, 10 mmol / L MgCl2). This process is repeated 3 times, and the volume is adjusted to OD. 600=0.5, the bacterial suspension was activated in the dark at 28℃ for 2-3 hours. Four-week-old, well-grown *Nicotiana benthamiana* seedlings were selected, and the activated *Agrobacterium* suspension was injected into the back of the tobacco plants using a 1 mL syringe (without the needle). After injection, the plants were cultured for another 2-3 days. Leaf tissue was then cut, and sections were prepared with the underside of the leaves facing upwards. The fluorescence expression and localization information of the leaves were observed using a confocal laser microscope. In this study, EGFP tags were used, with an excitation wavelength range of 488 nm and an image acquisition wavelength range of 515-540 nm. EGFP expression was observed using 488 nm excitation light, and images were saved.

[0097] The results are as follows Figure 3 As shown, under confocal laser microscopy, the fluorescent expression of the g1217 effector protein was observed on the cell membrane. It is speculated that this effector protein acts on the cell membrane and exerts its function on the cell.

Claims

1. The application of overexpression of the wheat dwarf smut effector protein gene g1217 in inhibiting BAX protein-induced necrosis of tobacco leaf tissue, characterized in that, The gene g1217 inhibits programmed cell death in tobacco plants induced by BAX protein; the nucleotide sequence of the gene g1217 is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the gene g1217 is shown in SEQ ID NO:

2.

3. The application of biomaterials overexpressing the *Ustilago maydis* effector protein gene g1217 in inhibiting BAX protein-induced necrosis of tobacco leaf tissue, characterized in that... The biomaterial inhibits BAX protein-induced programmed cell death in tobacco plants; the biomaterial is any one of A1) to A3) below: A1) An expression cassette containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1; A2) A recombinant vector containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1; A3) A recombinant microorganism containing a nucleic acid molecule with the sequence shown in SEQ ID NO:1, or a recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2), wherein the microorganism is Agrobacterium; The nucleotide sequence of the gene g1217 is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the gene g1217 is shown in SEQ ID NO:

2.

4. A method for inhibiting BAX protein-induced necrosis of tobacco leaf tissue, characterized in that, The method involves overexpressing the wheat dwarf smut effector protein gene g1217 in tobacco, wherein the gene g1217 inhibits programmed cell death in tobacco plants induced by BAX protein; the nucleotide sequence of the gene g1217 is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the gene g1217 is shown in SEQ ID NO:

2.

5. The method according to claim 4, characterized in that, The overexpression of the wheat dwarf smut effector protein gene g1217 in tobacco was achieved by transferring a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO:1 into Agrobacterium, introducing it into tobacco, and increasing the expression level of the g1217 gene in tobacco.

6. The method according to claim 5, characterized in that, The recombinant expression vector was constructed through the following steps: Step 1: Using cDNA synthesized by reverse transcription of total RNA extracted from wheat dwarf smut fungus as a template, PCR amplification was performed using primers g1217F1 and g1217R1 to obtain the g1217 gene fragment; the nucleotide sequence of primer g1217F1 is shown in SEQ ID NO:3, and the nucleotide sequence of primer g1217R1 is shown in SEQ ID NO:4; Step 2: Use restriction endonucleases Cla I and Sal I. The pGR107 vector was double-digested to recover the linearized pGR107 vector fragment; Step 3: Ligate the g1217 gene fragment from Step 1 with the linearized pGR107 vector fragment from Step 2, transform it into competent E. coli cells, and obtain positive clones through screening and sequencing verification, thus obtaining the recombinant expression vector pGR107-g1217.

7. The method according to claim 6, characterized in that, In step one, the PCR reaction system is as follows: 25 μL of 2×Master Mix, 2 μL each of 10 μM upstream and downstream primers, 0.5 μg of cDNA template, and ddH2O to 50 μL; the PCR reaction program is: 95℃ for 5 min; 95℃ for 1 min, 57℃ for 1 min, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

8. The method according to claim 5, characterized in that, The Agrobacterium is Agrobacterium tumefaciens GV3101.

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