Rice kernel smut fungus effector ThSCSP35 and application thereof in rice disease resistance

Abstract

The invention discloses a rice kernel smut effector factor ThSCSP35 and application thereof, and belongs to the technical field of genetic engineering. The effector factor ThSCSP35 is separated and cloned from the rice kernel smut, and the nucleotide sequence of the effector factor ThSCSP35 is shown as SEQ ID NO: 1. Through cloning and functional analysis of the rice kernel smut fungus effector factor ThSCSP35, the molecular mechanism of specific interaction and evolution between the rice kernel smut fungus microspecies and the rice variety is disclosed, so that the occurrence of the rice kernel smut fungus is effectively controlled.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a rice smut fungus effector ThSCSP_35 and its application in rice disease resistance. Background Technology

[0002] Rice grain smut is mainly caused by the rice grain smut fungus (Smut fungus). Tilletia horrida Caused by [unspecified pathogen], this is an important panicle disease. The pathogen infects rice grains, destroying the endosperm and transforming it into black, thick-walled spore masses, leading to reduced grain weight and severely degraded quality (producing black powder and off-flavors), causing significant economic losses. Especially in hybrid rice seed production fields, due to the characteristics of the female parent (usually a sterile line) such as necking, poor heading, and prolonged flowering period, its glume is more susceptible to the disease, often resulting in severe outbreaks.

[0003] In plant pathogen-microbe interaction research, "effectors" generally refer to a class of small-molecule proteins or other metabolites secreted by pathogens. Their core function is to be delivered into the host cell during infection, thereby inhibiting the host's defense response and promoting pathogen colonization and nutrient acquisition by interfering with the host's immune signaling pathways (PTI and ETI) and cellular physiological processes. However, there are few reports on the effectors of rice grain smut and their interaction with the host. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an effector factor ThSCSP_35 of rice smut fungus and its application. The present invention isolates and clones the effector factor ThSCSP_35 from rice smut fungus and analyzes the function of this gene, which helps to reveal the molecular mechanism of the specific interaction and evolution between rice smut fungus races and rice varieties, thereby effectively controlling the occurrence of rice smut.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a rice grain smut fungus effector ThSCSP_35 is provided, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0006] The amino acid sequence of the protein encoded by the aforementioned effector ThSCSP_35 is shown in SEQ ID NO: 2.

[0007] This should be understood as meaning that, without affecting protein activity, those skilled in the art can substitute, add, and / or delete one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 to obtain an amino acid sequence with equivalent function.

[0008] In addition, considering the degeneracy of codons, for example, the gene sequence encoding the above-mentioned protein can be modified in its coding region without changing the amino acid sequence, or in its non-coding region without affecting protein expression, therefore, the present application also includes the amino acid sequence with the same function formed by replacing, adding and / or deleting one or more amino acid residues in the gene sequence encoding the above-mentioned protein.

[0009] An expression vector comprising the above-mentioned effector ThSCSP_35.

[0010] A host cell comprising the above-mentioned effector ThSCSP_35 or an expression vector containing the above-mentioned effector ThSCSP_35.

[0011] A kit comprising the above-mentioned effector ThSCSP_35 or the protein encoded thereby or an expression vector or host cell containing the effector ThSCSP_35.

[0012] The above-mentioned rice smut effector ThSCSP_35 or the protein encoded thereby in eliciting plant immunity, improving plant resistance to rice smut, breeding transgenic plants resistant to rice smut, products for plant resistance to rice smut, designing molecular targets for pesticides, and rice breeding for disease resistance.

[0013] Further, the pathway for eliciting plant immunity is selected from at least one of the following ①-③: ① inducing the expression of resistance genes; ② inducing the accumulation of H2O2; ③ inducing the deposition of callose.

[0014] A reagent for preventing and treating rice smut, comprising the protein encoded by the above-mentioned effector ThSCSP_35.

[0015] A method for plant resistance to rice smut, comprising the following steps: Reducing the virulence of the rice smut fungus by interfering with the expression of the effector ThSCSP_35.

[0016] The application provides a rice smut effector ThSCSP_35 and an application thereof, and has the following beneficial effects: the application helps to reveal the molecular mechanism of the specificity interaction and evolution between the race of the rice smut and the rice variety by cloning and function analysis of the rice smut effector ThSCSP_35, and provides a basis for further exploring the interaction mechanism between the rice smut and the host. In practice, the molecular target of a new pesticide can be designed according to the structure and function of the gene; the receptor protein gene of the effector in the host cells such as rice can be knocked out or mutated to obtain a persistent disease-resistant variety; the application helps to establish a molecular detection system of the pathogenicity variation of the natural population of the rice smut, research the distribution of the effector of the rice smut in the natural population in the field, and reveal the composition and variation characteristics of the race in the population of the rice smut; and the application also helps to identify the disease resistance of the rice variety, reasonably arrange and replace the rice variety, and provide a reference for the green prevention and control of the rice smut. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The necrosis of epidermal cells is caused by the transient expression of the effector ThSCSP_35 cloned from different strains of the rice smut in tobacco leaves.

[0018] Figure 2 The signal peptide sequence (ThSCSP_35 SP ) of the rice smut effector ThSCSP_35 is verified to have a secretion function.

[0019] Figure 3 The signal peptide deletion sequence (ThSCSP_35ΔSP) of ThSCSP_35 transiently expressed in tobacco no longer causes the necrosis phenotype of leaf epidermal cells.

[0020] Figure 4 The expression level of ThSCSP_35 when the rice smut infects rice for 8h, 24h and 72h.

[0021] Figure 5 The detection results of the immune response of tobacco leaves activated by the rice smut effector ThSCSP_35; wherein A: the disease resistance related genes ERF1, LOX, PR2b, PR4a and RbohB are induced and up-regulated after the transient expression of ThSCSP_35 in N. benthamiana; B: ThSCSP_35 induces the accumulation of H2O2 and the deposition of callose in tobacco.

[0022] Figure 6 The subcellular localization of the rice smut effector ThSCSP_35 in N. benthamiana.

[0023] Figure 7To interfere with the expression of ThSCSP_35 and reduce the virulence test results of rice smut fungus strain JY-521. Detailed Implementation

[0024] The fungi genus *Smuts* have a wide host range, including rice, corn, sugarcane, sorghum, barley, and many other crops. During infection, *Smuts* secrete various effector factors that regulate the host's innate immunity and promote self-infection. These effector factors are key pathogenic factors ensuring the pathogen's infection of the host. However, research on the effector factors of rice grain smut and their interaction with the host is limited. Therefore, research on these effector factors not only lays a theoretical foundation for elucidating the molecular mechanisms of the interaction between *Smuts* and the host, but also facilitates the discovery of rice grain smut resistance gene resources by identifying target proteins in the host that recognize *Smuts* effector factors, further improving the smut resistance of mainstream rice male-sterile lines in production. Furthermore, based on the sequences and structures of *Smuts* effector factors, new pesticide targets can be designed, ultimately achieving effective control of rice grain smut and increasing agricultural yields and income.

[0025] This invention isolates and clones the effector factor ThSCSP_35 from rice smut fungus and analyzes the function of this gene. Based on the structure and function of this gene, pesticide molecular targets can be designed. In addition, it can also be applied to improve rice disease resistance breeding. For example, the gene sequence can be ligated into any transformation vector containing a fluorescent protein gene, and the effector factor and fluorescent protein can be covalently introduced into rice or other plant cells using any transformation method. Fluorescence confocal transmission electron microscopy can be used to observe the migration and localization of the effector factor expressed with the fluorescent protein in rice or other plant cells. This effector protein can be used as a bait protein to fish out the receptor protein that binds to the effector protein in rice or other plants. Genetic engineering methods can be used to knock out the receptor gene in rice or other plant cells or delete, add, or mutate one or more bases of the receptor gene to delete or change the function of the receptor gene, thereby obtaining resistant plants against a certain effector factor.

[0026] Furthermore, specific molecular markers can be generated based on this gene sequence information, including but not limited to SNP (single nucleotide polymorphism), SSR (simple sequence repeat polymorphism), RFLP (restriction endonuclease length polymorphism), and CAP (cutting amplified fragment polypeptide). These markers can be used to detect the dynamic changes in the physiological races and genetic structure of rice grain smut populations in the field, as well as the distribution of this effector in natural field populations. This helps in the identification of disease resistance in rice varieties and the identification of rice grain smut pathogen races, and also helps in the rational layout and rotation of disease-resistant varieties, so as to effectively control the occurrence of rice grain smut.

[0027] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1: Cloning of ThSCSP_35, an effector factor of rice grain smut. The rice smut fungus strain JY-521, isolated from rice grain smut samples, was transformed into PSA solid medium for activation. After 5 days, single colonies were picked and cultured in 50 mL of PSA liquid medium at 28°C and 200 rpm for 5 days. Mycelia were then collected by filtration through four layers of gauze, and total RNA was extracted by grinding with liquid nitrogen and reverse transcribed to obtain cDNA. Primers were designed based on the previous rice grain smut genome sequencing sequence, and the primer sequences are as follows: Forward primer: 5'-gacctcgactctagaggatccATGCGCTTCAACTTTCTTGCC-3' (SEQ ID NO: 3).

[0029] Reverse primer: 5'-gtccttgtagtcagaaggcctTCAGCGGCTGTGGCTGAT-3' (SEQ ID NO: 4).

[0030] PCR amplification was performed using cDNA obtained from reverse transcription as a template. The PCR reaction system (50µL) consisted of: 2µL cDNA template, 2µL each of forward and reverse primers, 10µL FastPfu Fly Buffer, 5µL 2.5mM dNTPs, 1µL FastPfu Fly high-fidelity enzyme, and 28µL ddH2O. The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 50 s, 55℃ annealing for 50 s, and 72℃ extension for 1 min, for a total of 35 cycles; followed by a final extension at 72℃ for 10 min. After the reaction, the obtained PCR product was sent to Qingke Xinyue Biotechnology Co., Ltd. (Chengdu) for sequencing. The sequencing results are as follows: ATGCGCTTCAACTTTCTTGCCACTTTGTCCGTCTTGACGGCCGGCACTGCCCTGGCCGCCAGCCTCGAGAGTCGCTCAGCATGCACTAGCGTCTGTGAAAACGCTAGTCTGAAGGGCACTATGTTGACTCGATGCCTGGTCAAGTGCGAATACTGCAGTGAAGCGGGGTCAAAGGGCTACATTCCTCCCTCCCG AGTCAACTGCTCCAACTACTGCGGCACGAACCGATCCTGCTACGACAACTGCAACGCCGCTCAGGCAAACTGCTGGCCACTCGGCAAGGGAACCACTTATGCATGCCCGGATGTCGGATACATGTGCACTTACTTGTGCAACAAGCAGGACCCCGTCGACCCTAGCATCTGCATCAGCCACAGCCGCTGA(SEQ ID No:1).

[0031] The amino acid sequence obtained through translation is shown below: MRFNFLATLSVLTAGTALAASLESRSACTSVCENASLKGTMLTRCLVKCEYCSEAAGSKGYIPPSRVNCSNYCGTNRSCYDNCNAAQANCWPLGKGTTYACPDVGYMCTYLCNKQDPVDPSICISHSR (SEQ ID No: 2).

[0032] Example 2: Construction and transformation of ThSCSP_35 prokaryotic expression vector The PCR-amplified target gene fragment was recovered using an agarose gel DNA recovery kit (OMEGA, USA). Following the pEASY-Blunt Elkit instructions, the target gene was ligated into the expression vector 35S-PMDC32. 2 μL of the ligation product was mixed with 50 μL of LDH5a competent cells (CWO808S, CWBIO, Beijing) for transformation. The transformed plasmid was plated on YEP solid medium containing kanamycin (50 mg / L) and rifampin (50 mg / L) for selective selection, yielding the recombinant plasmid. Sequencing revealed that the gene ThSCSP_35 was identical to the genome sequence, as shown in SEQ ID NO:3. Electroporation of GV3101 competent cells, followed by rifampin and kanamycin resistance selection, yielded an Agrobacterium strain carrying the transient expression vector.

[0033] Example 3: Transient expression of ThSCSP_35 in Benedictine leaves Single clones of Agrobacterium carrying the target gene were streaked to isolate the bacteria. Each clone was picked and placed in YEP medium containing rifampicin (50 mg / L) and kanamycin (50 mg / L), and incubated at 28°C for 16 h. After centrifugation, the bacterial cells were collected and resuspended in MES resuspension [10 mM MES (pH 5.6), 10 mM MgCl2, and 150 µM acetylsyl syringone], and the OD600 was adjusted to 0.6. The cells were then incubated at room temperature in the dark for 3 h. Tobacco leaves were injected using a syringe, and the condition of the tobacco leaves was observed. Results are shown in […]. Figure 1 .Depend on Figure 1 It can be seen that the negative control (Agrobacterium tumefaciens liquid carrying the GFP protein expression vector) did not cause cell necrosis in the tobacco leaves after injection, while the positive control (Agrobacterium tumefaciens liquid carrying the mouse apoptosis protein BAX) and the Agrobacterium tumefaciens liquid carrying the target gene ThSCSP_35 showed obvious cell necrosis after injection into the tobacco leaves 4 days later.

[0034] Example 4: Validation of the signal peptide secretion function of ThSCSP_35 To clarify the signal peptide of ThSCSP_35 (ThSCSP_35 SP To determine if ThSCSP_35 possesses secretory function, primer sequences were designed: forward primer: 5'-cggaattttaattaagaattcATGCGCTTCAACTTTCTTGCC-3' (SEQ ID NO:5); reverse primer: 5'-cactatagggagaacctcgagGGCCAGGGCAGTGCCGGC-3' (SEQ ID NO:6). ThSCSP_35 was cloned. SP , further ThSCSP_35 SP The segment was constructed on the pSUC2 vector, using rice blast fungus (which lacks secretory function) Magnaporthe oryzae The signal peptide sequence of the Mg87 protein (Mg87) SP ) was used as a negative control, with the secretory pathogen of soybean Phytophthora ( ) Phytophthora sojae The signal peptide sequence of the Avr1b protein (Avr1b) SP () served as a positive control for functional verification; results are shown below. Figure 2 .Depend on Figure 2 As shown in Figure A, all strains grew well on CMD-W culture, indicating successful plasmid transformation into the YTK12 yeast strain. The positive control Avr1b signal peptide guides the secretion of fructosylase, thus allowing it to grow on YPRAA medium containing raffinose. ThSCSP_35 can be observed... SP Similar to the positive control, the ability of fructosylase-deficient yeast to grow on YPRAA medium was restored. However, the Mg87 signal peptide of the negative control could not grow on YPRAA plates. Figure 2A). In addition, the enzyme activity of secreted invertase in the culture medium was also tested. The results showed that the ThSCSP_35 signal peptide, like the Avr1b signal peptide, has the function of secreting invertase into the culture medium, converting 2,3,5-triphenyltetrazolium chloride (TTC) into insoluble red 1,3,5-triphenylmethyl (TPF), while the non-secreting Mg87 signal peptide did not change color. Figure 2 B). The yeast sucrose experiment and the TTC experiment together verified ThSCSP_35. SP It can perform its secretory function normally.

[0035] Example 5: Effect of the ThSCSP_35 signal peptide on its ability to induce epidermal cell necrosis in tobacco leaves To clarify the effect of the ThSCSP_35 signal peptide on its ability to induce epidermal cell necrosis in tobacco leaves, primer sequences were designed: forward primer: 5'-gacctcgactctagaggatccATGGCCAGCCTCGAGAGTCG-3' (SEQ ID NO:7); reverse primer: 5'-gtccttgtagtcagaaggcctTCAGCGGCTGTGGCTGAT-3' (SEQ ID NO:7). NO:8) The deleted signal peptide sequence of ThSCSP_35 (ThSCSP_35ΔSP) was cloned, and the ThSCSP_35ΔSP sequence was constructed into the 35S-PMDC32 vector (a recombinant expression vector constructed by inserting a 35S promoter in front of the multiple cloning site of the basic vector PMDC32) using T4 ligase. After confirming the correct sequence by transforming it into E. coli, it was then transformed into Agrobacterium GV3101 for transient expression in tobacco. The presence of ThSCSP_35ΔSP in tobacco leaf epidermal cells was observed 3-5 days after injection. Figure 3 It can be seen that necrosis was observed at sites where positive controls Bax and ThSCSP_35 were injected, while no necrosis was observed at sites where negative controls GFP and ThSCSP_35ΔSP were injected. Figure 3 This study confirmed that the signal peptide has a significant effect on the ThSCSP_35-induced epidermal cell necrosis function in tobacco leaves.

[0036] Example 6: Expression pattern of ThSCSP_35 during infection by rice grain smut. To determine whether ThSCSP_35 is induced during rice smut infection, we used quantitative real-time PCR (qRT-PCR) to detect the expression level of the ThSCSP_35 gene at different infection time points (0, 8, 24, and 72 h). Total RNA was extracted from rice panicles infected at 8, 24, and 72 h, and cDNA was obtained by reverse transcription. Using a LightCycler 96 quantitative PCR instrument, with the reverse-transcribed cDNA as a template and the UBQ gene as an internal control, PCR amplification was performed using the TB Green (TaKaRa) dye method. Each reaction was biologically replicated three times. After the reaction, the expression levels were determined according to the 2... -△△Ct The relative expression levels of genes at different time points in rice infected with rice smut were analyzed.

[0037] The results are as follows Figure 4 As shown, ThSCSP_35 was induced to be upregulated during the infection of the rice grain smut fungus. These results indicate that ThSCSP_35 is upregulated in the early stage of rice grain smut infection and plays an important role in the infection process. The primer sequences used for qRT-PCR are as follows: UBQ: Forward primer: 5'-ACCACTTCGACCGCCACTACT-3' (SEQ ID NO:9).

[0038] Reverse primer: 5'-ACGCCTAAGCCTGCTGGTT-3' (SEQ ID NO:10).

[0039] ThSCSP_35: Forward primer: 5'-ACATTCCTCCCTCCCGAGTCAAC-3' (SEQ ID NO:11).

[0040] Reverse primer: 5'-CATAAGTGGTTCCCTTGCCGAGTG-3' (SEQ ID NO:12).

[0041] Example 7: Detection of ThSCSP_35-induced expression of resistance-related genes in tobacco leaves To investigate whether the effector ThSCSP_35 elicits an immune response in tobacco, we extracted RNA from tobacco leaves after transient expression of ThSCSP_35 for 12 hours, reverse transcribed it into cDNA, and used qRT-PCR to detect the expression levels of key genes in the disease resistance pathway: RbohB, ERF1, LOX, PR4a, and PR2b. Using transient GFP expression as a control, we employed a LightCycler 96 quantitative PCR instrument, using the reverse-transcribed cDNA as a template and the Actin gene as an internal control, and performed PCR amplification using TB Green (TaKaRa) dye. Each reaction was biologically replicated three times. After the reaction, the results were analyzed according to the 2... -△△Ct The relative expression levels of five resistance-related genes were analyzed using [method name missing], and the results are shown in [see attached image]. Figure 5 .Depend on Figure 5 As shown in Figure A, transient expression of ThSCSP_35 in tobacco leaves significantly activated the expression of genes related to disease resistance pathways. The primer sequences used for qRT-PCR are as follows: Nb-Actin: Forward primer: 5'-TGGCATCTCTCAGCACATTCC-3' (SEQ ID NO:13).

[0042] Reverse primer: 5'-TGCACAATGGATGGGCCAGA-3' (SEQ ID NO:14).

[0043] Nb-ERF1: Forward primer: 5'-GCTCTTAACGTCGGATGGTC-3' (SEQ ID NO:15).

[0044] Reverse primer: 5'-AGCCAAACCCTAGCTCCATT-3' (SEQ ID NO:16).

[0045] Nb-LOX: Forward primer: 5'-AAAACCTATGCCTCAAGAAC-3' (SEQ ID NO:17).

[0046] Reverse primer: 5'-ACTGCTGCATAGGCTTTGG-3' (SEQ ID NO:18).

[0047] Nb-RbohB: Forward primer: 5'-TCACAAGAGCTCAGGCGTTT-3' (SEQ ID NO:19).

[0048] Reverse primer: 5'-TCATCGAACCGCTTCTCGAC-3' (SEQ ID NO:20).

[0049] Nb-PR2b: Forward primer: 5'-AGGTGTTTGCTATGGAATGC-3' (SEQ ID NO:21).

[0050] Reverse primer: 5'-TCTGTACCCACCATCTTGC-3' (SEQ ID NO:22).

[0051] Nb-PR4a: Forward primer: 5'-CAACCCACAGAACATTAACTGG-3' (SEQ ID NO:23).

[0052] Reverse primer: 5'-TTGTCGGCATCCCAAGTAGT-3' (SEQ ID NO:24).

[0053] Example 8: Detection of H2O2 accumulation in tobacco leaves induced by ThSCSP_35 (1) After ThSCSP_35 was transiently expressed in tobacco leaves for 48 h, it was placed in 1 mg / mL DAB staining solution and incubated at 25 °C for 10 h. (2) Remove the leaves and transfer them to a boiling water bath in 95% ethanol for 15 minutes until the leaves turn colorless; (3) Take out the leaves and rinse them twice with distilled water, then transfer them to a 2.5 g / mL chloral hydrate solution until the background is completely eliminated; (4) Take out the sample, wash it twice with distilled water and take a picture.

[0054] Depend on Figure 5 B indicates that transient expression of ThSCSP_35 in tobacco leaves induces the accumulation of H2O2.

[0055] Example 9: Detection of Tobacco Callose Deposition Induced by ThSCSP_35 (1) Take tobacco leaves that have grown for about 3-4 weeks and inject them with ddH2O and Agrobacterium tumefaciens solution carrying the target gene ThSCSP_35 respectively; (2) After 24 hours of induction, remove the treated tobacco leaves and put them into Callose decolorizing solution. Place them in a 65°C oven and heat for 1-2 hours. Shake 2-3 times during this period to ensure complete decolorization. Avoid light. (3) After the chlorophyll in the tobacco leaves is completely removed, discard the decolorizing solution and wash them 1-2 times with an appropriate volume of 50% ethanol and ddH2O respectively. (4) Immerse the tobacco leaves in Callose staining solution for 30-60 minutes; (5) Take out the tobacco leaves and spread them flat on a glass slide. Add an appropriate amount of 50% glycerol and examine the callosity using ultraviolet light. Callose destaining solution: 80 mL lactic acid, 80 mL glycerol, 80 mL ddH2O, 80 mL phenol and 640 mL ethanol; Callose staining solution: 17.1165 g K2HPO4∙3H2O was added to 500 mL ddH2O, the pH was adjusted to 9.5, 0.05 g aniline blue was added, and the solution was stored in the dark.

[0056] Depend on Figure 5 B indicates that transient expression of ThSCSP_35 in tobacco leaves induces callose deposition.

[0057] Example 10: Subcellular localization of ThSCSP_35 The amplified ThSCSP_35ΔSP sequence was constructed into the PHB-YFP vector using T4 ligase. The successfully ligated PHB-ThSCSP_35ΔSP-YFP vector was transformed into Agrobacterium GV3101, injected into tobacco, and observed and photographed using confocal microscopy. The specific steps are as follows: (1) Take 0.1-0.5µL of DNA and 10-20µL of competent cells (GV3101) and add them to a 1.5mL EP tube, then mix well; (2) Place on ice for 30 min, then in liquid nitrogen for 5 min; (3) Quickly remove it from the liquid nitrogen and place it in a 37°C water bath for 5 minutes; (4) Add 200-400µL of LB liquid antibiotic-free medium, gently shake to mix, and incubate at 28℃ and 200rpm for 4-5h until OD600=1.0; (5) Centrifuge (4000 rpm) for 10 min, discard the supernatant, and keep about 200 µL of supernatant. Gently mix it by pipetting with a pipette tip. (6) Spread on the corresponding antibiotic culture dish and incubate in a constant temperature incubator at 28℃ for 2 days; (7) Plaque PCR identification; (8) Preserve and extract plasmids from the correctly tested positive clones by shaking. (9) When injecting tobacco, take 5 µL of the positive clones that have been successfully transformed by Agrobacterium into 1 mL of the corresponding LB liquid medium and culture for 2-3 days; (10) Pipette 600µL of the culture medium into a 2mL EP tube, centrifuge (6000rpm) for 5min, and discard the supernatant; (11) Add 1.5 mL of pre-prepared MMA Buffer (10 mM MgCl2, 10 mM MES pH=5.8 and 100 μMAS), gently mix with a pipette tip, adjust to OD600=0.7-1.0, and place at room temperature in the dark for 3-4 hours; (12) Use a syringe to draw up the bacterial solution and gradually push the bacterial solution into the lower epidermal cells of the leaf, and mark it with a pen or label. (13) The injected tobacco plants were cultured in the dark for 12 hours, then placed in a normal incubator, and observed and photographed 2 days later.

[0058] The primers used for amplifying the ThSCSP_35ΔSP sequence are as follows: Forward primer: 5'-accagtctctctctcaagcttATGGCCAGCCTCGAGAGTCG-3' (SEQ ID NO: 25).

[0059] Reverse primer: 5'-gctcaccatactagtggatccTCAGCGGCTGTGGCTGAT-3' (SEQ ID NO: 26).

[0060] The results are as follows Figure 6 The results showed that the PHB-YFP vector (control) was expressed in both the cell nucleus and cell membrane, while the fluorescence localization excited by the expression of the ThSCSP_35ΔSP fusion protein was mainly observed in the cell membrane. These results indicate that ThSCSP_35ΔSP is localized in the cell membrane in tobacco.

[0061] Example 11: Interference with ThSCSP_35 expression reduced the virulence of rice grain smut fungus strain JY-521. Based on the sequence information of the ThSCSP_35 gene, we designed short interfering RNAs (siRNAs) for this gene. The specific sequences are: 21nt guide: 5'-AGUAAGUGCACAUGUAUCCGA-3' (SEQ ID NO:27, with the U replaced by T during sequence listing, specifically: 5'-AGTAAGTGCACATGTATCCGA-3'); 21nt passenger: 5'-GGAUACAUGUGCACUUACUUG-3' (SEQ ID NO:28, with the U replaced by T during sequence listing, specifically: 5'-GGATACATGTGCACTTACTTG-3'). The ThSCSP_35 siRNAs were synthesized into the pBWA(V)HS expression vector, and the recombinant vector plasmid was transformed into Agrobacterium strain GV3101 (using the same method as in Example 2). Furthermore, Agrobacterium-mediated transformation was used to transfer ThSCSP_35 siRNAs into rice 9311B. The specific steps are as follows: (1) After removing the hulls, disinfect the rice seeds with 75% alcohol for 1 min, disinfect with sodium hypochlorite for 20 s, wash with sterile water 3 times, place them on sterile filter paper and blow dry, sow them evenly on MS medium, and culture them at 24℃ in the dark for 8-10 days until the seeds germinate. Cut off fresh cotyledons for later use. (2) Activation of the successfully transformed Agrobacterium strain in LB solid medium; (3) Select a single colony of activated Agrobacterium and inoculate it into LB liquid medium. Shake and culture for 24 hours until the logarithmic growth phase. Soak the fresh cotyledons in (1) into the medium containing Agrobacterium for infection. (4) After removing the Agrobacterium tumefaciens bacterial solution, the infected cotyledon tissue was transferred to a co-culture medium and co-cultured for 2-3 days; (5) Collect the co-cultured callus into a culture flask, add carboxylic acid water for washing, and then dry the callus with sterile filter paper. (6) The dried callus tissue was inoculated onto the screening medium for screening culture; (7) The resistant callus selected and cultured is transferred to the differentiation medium for differentiation culture until green shoots appear in the callus tissue.

[0062] qRT-PCT confirmed that the expression of ThSCSP_35 was significantly inhibited 12 h after infection of the ThSCSP_35 RNAi line by the rice grain smut fungus JY-521 strain. Figure 7A). Inoculation of ThSCSP_35 interference lines (RNAi) and wild-type plants with the highly virulent strain JY-521 of rice grain smut fungus revealed that the introduction of ThSCSP_35 siRNAs weakened the susceptibility of rice plants. Figure 7 B).

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rice grain smut fungus effector ThSCSP_35, characterized in that, The nucleotide sequence of the effector ThSCSP_35 is shown in SEQ ID NO:

1.

2. The protein encoded by the rice grain smut effector ThSCSP_35 as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. An expression carrier, characterized in that, Includes the rice grain smut fungus effector ThSCSP_35 as described in claim 1.

4. A host cell, characterized in that, It includes the rice grain smut fungus effector ThSCSP_35 as described in claim 1 or the expression vector as described in claim 3.

5. A reagent kit, characterized in that, The protein encoded by the rice grain smut effector ThSCSP_35 as described in claim 1 or claim 2, or the expression vector as described in claim 3, or the host cell as described in claim 4.

6. The application of the rice grain smut effector ThSCSP_35 as described in claim 1 or the protein encoded by the rice grain smut effector ThSCSP_35 as described in claim 2 in stimulating plant immunity, improving plant resistance to rice grain smut, breeding transgenic plants resistant to rice grain smut, producing products resistant to rice grain smut, designing pesticide molecular targets, and breeding rice for disease resistance.

7. The application according to claim 6, characterized in that, The pathways for stimulating plant immunity are selected from at least one of the following ①-③: ① Inducing the expression of resistance genes; ②Induces the accumulation of H2O2; ③Induces the deposition of callosity.

8. A reagent for controlling rice grain smut, characterized in that, Includes the protein encoded by the rice grain smut effector ThSCSP_35 as described in claim 2.

9. A method for plant resistance to rice grain smut, characterized in that, Includes the following steps: The virulence of rice grain smut fungus was reduced by interfering with the expression of the effector ThSCSP_35.

Images