Rice kernel smut disease effector factor ThSCSP20 and application thereof

Abstract

The invention discloses a rice kernel smut fungus effector ThSCSP20 and application thereof, and belongs to the technical field of gene engineering. The nucleotide sequence of the effector ThSCSP20 is as shown in SEQ ID NO: 1. Through cloning of the rice kernel smut fungus effect factor ThSCSP20 and function analysis of the rice kernel smut fungus effect factor ThSCSP20 in plant immunity excitation, the specific interaction between the rice kernel smut fungus microspecies and the rice variety and the molecular mechanism of evolution of the specific interaction are disclosed, and a basis is provided for further exploration of the interaction mechanism of the rice kernel smut fungus and a host.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a rice smut effector ThSCSP_20 and application thereof. BACKGROUND

[0002] Rice is one of the important food crops in China, and its production directly affects the economic development of the Chinese nation. Hybrid rice has been planted in increasing areas year by year due to its high yield. Sterile lines are important germplasm resources for breeding high-yield hybrid rice, and Tilletia horrida Rice smut caused by the smut fungus mainly invades the floral organs of sterile lines, and the chlamydospore is the main form of overwintering in seeds and soil. In recent years, in order to improve the seed yield of hybrid rice, the angle of the stigma of the sterile line has been increased, and the rate of stigma exposure has been improved, which provides favorable host conditions for the infection of the pathogen, and has become one of the important diseases that restrict the seed production of hybrid rice and the propagation of sterile lines in China.

[0003] At present, due to the lack of sterile line germplasm resources resistant to rice smut, the strong stress resistance of chlamydospores, the difficulty in field inoculation identification, and the different disease investigation and classification standards, the research on the interaction between rice smut fungus and host is slow, and there is still no systematic method for its prevention and control. In agricultural production, chemical pesticides are mainly used to prevent and control rice smut, but the use of chemical pesticides has brought environmental problems and the problem of pathogen resistance. The smut fungus has a wide host range, including rice, corn, sugarcane, sorghum, barley and other crops. The smut fungus can secrete various effectors to regulate the innate immunity of the host and promote its own infection during the process of infecting the host. These effectors are key pathogenic factors that ensure the infection of the pathogen to the host. However, there are few reports on the effectors of rice smut and their interaction with the host. SUMMARY

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a rice smut effector ThSCSP_20 and application thereof. The effector ThSCSP_20 is isolated and cloned from the rice smut fungus, and the function of the gene is analyzed, which helps to reveal the molecular mechanism of the specificity interaction between the race of rice smut fungus and the rice variety and its evolution, and thus effectively controls the occurrence of rice smut.

[0005] The technical scheme for solving the above-mentioned technical problems is as follows: a rice smut effector ThSCSP_20 is provided, and the nucleotide sequence of the effector is shown in SEQ ID NO: 1.

[0006] The protein encoded by the above-mentioned effector ThSCSP_20 has an amino acid sequence shown in SEQ ID NO: 2.

[0007] It should be understood herein that one or more amino acids are added, substituted and / or deleted from the amino acid sequence shown in SEQ ID NO: 2 by those skilled in the art without affecting the activity of the protein, 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_20.

[0010] A host cell comprising the above-mentioned effector ThSCSP_20 or an expression vector containing the above-mentioned effector ThSCSP_20.

[0011] A kit comprising the above-mentioned effector ThSCSP_20 or a protein encoded thereby or an expression vector or host cell containing the above-mentioned effector ThSCSP_20.

[0012] The above-mentioned Ustilaginoidea virens effector ThSCSP_20 or a protein encoded thereby in eliciting plant immunity, improving plant resistance to Ustilaginoidea virens, cultivating transgenic plants resistant to Ustilaginoidea virens, products for plant resistance to Ustilaginoidea virens, and designing molecular targets of pesticides.

[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 Ustilaginoidea virens, comprising a protein encoded by the above-mentioned effector ThSCSP_20.

[0015] The application provides a rice smut pathogenicity factor ThSCSP_20 and an application thereof, and has the following beneficial effects: the application helps to reveal the molecular mechanism of the specificity interaction between the race of the rice smut pathogenicity factor and the rice variety and the evolution thereof, and provides a basis for further exploring the interaction mechanism between the rice smut pathogenicity factor and the host. In practice, the gene can be introduced into a disease susceptible rice sterile line material by combining molecular biology technology and field hybridization to obtain a rice sterile line material with high outcrossing resistance to the rice smut; and the application also helps to establish a molecular detection system for the pathogenicity variation of the natural population of the rice smut, research the distribution of the pathogenicity factor in the natural population in the field, reveal the composition and variation characteristics of the race in the population of the rice smut, and provide a reference for the green prevention and control of the rice smut. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The rice smut pathogenicity factor ThSCSP_20 is transiently expressed in tobacco leaves to cause a necrotic reaction of epidermal cells.

[0017] Figure 2 The signal peptide sequence (ThSCSP_20-SP) of the rice smut pathogenicity factor ThSCSP_20 is verified for secretion function, and the deletion of the signal peptide sequence (ThSCSP_20-SP) of ThSCSP_20 does not cause a necrotic phenotype of leaf epidermal cells in tobacco. SP

[0018] Figure 3 RT-PCR is used to detect the expression of the rice smut pathogenicity factor ThSCSP_20 at different infection time points.

[0019] Figure 4 The detection results of the rice smut pathogenicity factor ThSCSP_20 activating the immune response of tobacco leaves; wherein a: after transient expression of ThSCSP_20 in N. benthamiana, the disease resistance related genes ERF1, LOX and PR2b are induced and up-regulated; b: ThSCSP_20 induces the accumulation of H2O2 in tobacco; c: ThSCSP_20 induces the deposition of callose in tobacco.

[0020] Figure 5 The subcellular localization of the rice smut pathogenicity factor ThSCSP_20 in N. benthamiana. DETAILED DESCRIPTION

[0021] ​Ustilago spp. can infect a wide range of hosts, including rice, maize, sugarcane, sorghum, barley and other crops. In production, chemical pesticides are mainly used to control smut, while traditional fungicides and insecticides have problems such as environmental pollution, pesticide residues and easy resistance. In recent years, research has shown that plant resistance elicitors have become a powerful alternative to traditional fungicides and insecticides in sustainable agriculture. At present, a number of plant resistance elicitors including proteins, sugars, lipids and peptides have been isolated from a variety of plant pathogens and biocontrol microorganisms. However, there are few reports on the elicitors of rice smut and their breeding applications. Therefore, the study of effectors not only lays a theoretical foundation for elucidating the molecular mechanism of interaction between rice smut and host, but also promotes the mining of rice smut resistance gene resources by identifying the target proteins in the host that recognize the effectors of rice smut, further improving the smut resistance of mainstream rice sterile lines in production; In addition, based on the sequence and structure of the effectors of rice smut, new biological pesticides can be designed to effectively control rice smut and increase agricultural production and income.

[0022] The effector ThSCSP_20 is isolated and cloned from Ustilaginoidea virens, which can be introduced into susceptible rice sterile line materials by field crossing to breed high outcrossing rice sterile line materials with resistance to rice smut and improve hybrid rice seed production; The gene sequence can be linked to any transformation vector containing a fluorescent protein gene, and the effector and fluorescent protein can be covalently introduced into rice or other plant cells by any transformation method. The migration and localization of the effector fused with the fluorescent protein in the plant cells can be observed by fluorescence confocal transmission electron microscopy. The effector protein is used as a bait protein to fish out the receptor protein in rice or other plants that binds to the effector protein, and the rice smut resistance / susceptibility gene is obtained. Further, the receptor gene in rice or other plant cells is overexpressed / knocked out by genetic engineering method to obtain stable resistant plants.

[0023] In addition, specific molecular markers can be generated according to the sequence information of the gene, including but not limited to SNP (single nucleotide polymorphism), SSR (simple sequence repeat polymorphism), RFLP (restriction enzyme length polymorphism) and CAP (cleavage amplification fragment polypeptide). These markers can be used to detect the physiological race of rice smut population in the field and the dynamic changes of its genetic structure, as well as the distribution of the effector in the natural population in the field, which is helpful for the identification of disease resistance of rice varieties and the identification of smut races, so as to effectively control the occurrence of rice smut.

[0024] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, conventional conditions or manufacturer's recommended conditions are used in the examples. Unless otherwise indicated, the reagents or instruments used are conventional products available commercially.

[0025] Example 1: Cloning of the S. oryzae effector ThSCSP_20 The S. oryzae strain JY-521 isolated from S. oryzae samples was transformed into PSA solid medium for activation, and after 5 days, single colonies were picked into 50 mL of PSA liquid medium and cultured at 28°C, 200 r / min for 5 days, then filtered with four layers of gauze to collect mycelium, and total RNA was extracted by grinding with liquid nitrogen, and cDNA was obtained by reverse transcription. Primers were designed according to the previous S. oryzae genome sequencing sequence, and the primer sequences were as follows: Forward primer: 5'-gacctcgactctagaggatccATGCAACGCTCCGTCTTCG-3' (SEQ ID NO: 3).

[0026] Reverse primer: 5'-gtccttgtagtcagaaggcctTTAAGCCCGGGTAATCTTGTAATG-3' (SEQ ID NO: 4).

[0027] PCR amplification was performed using the reverse-transcribed cDNA as the template, and the PCR reaction system was (50 µL): cDNA template 2 µL, forward and reverse primers 2 µL each, FastPfu Fly Buffer 10 µL, 2.5 mM dNTPs 5 µL, FastPfu Fly high-fidelity enzyme 1 µL, ddH2O 28 µL; the PCR amplification program was: 94°C pre-denaturation for 4 min; 94°C denaturation for 50 s, 55°C annealing for 50 s, 72°C extension for 1 min, a total of 35 cycles; 72°C post-extension for 10 min. After the reaction, the obtained PCR product was sent to Chengke New Industry Biotechnology Co., Ltd. (Chengdu) for sequencing, and the sequencing results were as follows: ATGCAACGCTCCGTCTTCGCATCCGCCGCGCTCGCCGCCTCCGCCTTTCTCATCGGCGCAAACGCGCTTCCAGCTACCAGCTCTGCCTCAGCAACCGTCTCTTGCAGCTCCACTCCACTCTGGTCCAACCACGGATTGAACATCACCGAGCTCCTCGGCCACTCCAAGCCAGGCGTCATTGCCACCGGCACCAATCACAACTCCCACGATCGTCTGCTGGCCTACCGAAACGCAACCGAGTACACATCCCAATGGGCATTCCACGTCTGCCAATTTGACGGTGTTCCCAAGACCAAGTCAAGCGATGACGTTGTGTACGGTCGCCTCAAAATTTACGATGAGGGATGCGCTGTTGTGGATGCGTATCCCAAGCTTGATTCGGGCTCATCTAAAGAGATCCAGTTTATCGGTGACTCGGCGAGCAAGAAATCAACAGGCGTCTACGTCTTTGCGATCCACGAGGATGACTATCTGCCCAAGTTCCCACTGAAGGAAGTTGCGATTCGGAAGGCGGACACGTTCAGCTCGAAGCATTACAAGATTACCCGGGCTTAA (SEQ ID NO: 1).

[0028] The amino acid sequence obtained by translation is shown below: MQRSVFASAALAASAFLIGANALPATSSASATVSCSSTPLWSNHGLNITELLGHSKPGVIATGTNHNSHDRLLAYRNATEYTSQWAFHVCQFDGVPKTKSSDDVVYGRLKIYDEGCAVVDAYPKLDSGSSKEIQFIGDSASKKSTGVYVFAIHEDDYLPKFPLKEVAIRKADTFSSKHYKITRA (SEQ ID NO: 2).

[0029] Example 2: Construction and transformation of ThSCSP_20 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_20 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.

[0030] Example 3: Transient expression of ThSCSP_20 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_20 showed obvious cell necrosis after injection into the tobacco leaves 4 days later.

[0031] Example 4: Validation of the signal peptide secretion function of ThSCSP_20 To clarify the signal peptide of ThSCSP_20 (ThSCSP_20 SP To determine whether ThSCSP_20 possesses secretory function, primer sequences were designed: forward primer: 5'-cggaattttaattaagaattcATGCAACGCTCCGTCTTCG-3' (SEQ ID NO:5); reverse primer: 5'-cactatagggagaacctcgagCGCGTTTGCGCCGATGAG-3' (SEQ ID NO:6). The cloned ThSCSP_20... SP , further ThSCSP_20 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_20 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 2 a). In addition, the enzyme activity of secreted invertase in the culture medium was also tested. The results showed that the ThSCSP_20 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 assay and the TTC assay together validated ThSCSP_20. SP It can perform its secretory function normally.

[0032] Example 5: Effect of the ThSCSP_20 signal peptide on its ability to induce epidermal cell necrosis in tobacco leaves To determine the effect of the signal peptide of ThSCSP_20 on its function of inducing necrosis of epidermal cells of tobacco leaves, the following primer sequences were designed: forward primer: 5'-gacctcgactctagaggatccATGCTTCCAGCTACCAGCTCTG-3' (SEQ ID NO: 7); reverse primer: 5'-gtccttgtagtcagaaggcctTTAAGCCCGGGTAATCTTGTAATG-3' (SEQ ID NO: 8) to clone the sequence of ThSCSP_20 with the signal peptide deleted (ThSCSP_20-SP). The sequence of ThSCSP_20-SP was constructed in a 35S-PMDC32 vector (a recombinant expression vector constructed by inserting a 35S promoter in front of the multiple cloning site of a basic vector PMDC32) using T4 ligase, and then introduced into E. coli to determine the sequence, and then introduced into Agrobacterium GV3101, and then used for transient expression in tobacco. Necrosis of epidermal cells of tobacco leaves induced by ThSCSP_20-SP was observed 3-5 days after injection. It was found from the graph in Figure 6C that necrosis was observed at the injection sites of the positive controls Bax and ThSCSP_20, but not at the injection sites of the negative controls GFP and ThSCSP_20-SP, indicating that the signal peptide had a significant effect on the function of ThSCSP_20 in inducing necrosis of epidermal cells of tobacco leaves. Figure 2

[0033] Example 6: Expression pattern of ThSCSP_20 during infection of rice by S. oryzae To determine whether ThSCSP_20 is induced to express during infection of rice by S. oryzae, the expression level of ThSCSP_20 at different infection time points (0, 24 and 72 h) was detected by real-time fluorescent quantitative PCR (qRT-PCR). Total RNA was extracted from the inflorescences of rice at 0, 24 and 72 h after infection, and cDNA was obtained by reverse transcription. The cDNA obtained by reverse transcription was used as a template, and the UBQ gene was used as an internal reference gene. PCR amplification was performed using the TB Green (TaKaRa) dye method, and each reaction was biologically repeated 3 times. After the reaction, the relative expression level of the gene at different time points during infection of rice by S. oryzae was analyzed according to the 2 -△△Ct method.

[0034] The results, as shown in Figure 6D, indicated that ThSCSP_20 was induced to express at an up-regulated level during infection of rice by S. oryzae, and played an important role in the process. Figure 3 The primer sequences used in qRT-PCR are as follows: UBQ: ​Forward primer: 5'- ACCACTTCGACCGCCACTACT-3' (SEQ ID NO: 9).

[0035] Reverse primer: 5'-ACGCCTAAGCCTGCTGGTT-3' (SEQ ID NO: 10).

[0036] ThSCSP_20: Forward primer: 5'- CGCCAACTGTTCTCCTACTCCAAG-3' (SEQ ID NO: 11).

[0037] Reverse primer: 5'-TCCGAATCGCAACTTCCTTCAGTG-3' (SEQ ID NO: 12).

[0038] Example 7: Detection of ThSCSP_20-induced expression of resistance-related genes in tobacco leaves To investigate whether the effector ThSCSP_20 triggers the immune response of tobacco, we extracted RNA from tobacco leaves 12 h after transient expression of ThSCSP_20 in tobacco leaves, reverse-transcribed it into cDNA, and detected the expression levels of key genes ERF1, LOX, PR4a, and PR2b in the disease resistance pathway using qRT-PCR. The GFP gene was used as a control. The LightCycler 96 quantitative PCR instrument was used, the reverse-transcribed cDNA was used as the template, the Actin gene was used as the internal reference gene, TB Green (TaKaRa) dye method was used for PCR amplification, and each reaction was biologically repeated 3 times. After the reaction, the relative expression amounts of 5 resistance-related genes were analyzed according to the 2 -△△Ct method, and the results are shown in Figure 4 a. It can be seen from Figure 4 a that the transient expression of ThSCSP_20 in tobacco leaves significantly activated the expression of genes related to the disease resistance pathway. The primer sequences used in qRT-PCR are as follows: Nb-Actin: Forward primer: 5'-TGGCATCTCTCAGCACATTCC-3' (SEQ ID NO: 13).

[0039] Reverse primer: 5'-TGCACAATGGATGGGCCAGA-3' (SEQ ID NO: 14).

[0040] Nb-ERF1: Forward primer: 5'-GCTCTTAACGTCGGATGGTC-3' (SEQ ID NO: 15).

[0041] Reverse primer: 5'-AGCCAAACCCTAGCTCCATT-3' (SEQ ID NO:16).

[0042] Nb-LOX: Forward primer: 5'-AAAACCTATGCCTCAAGAAC-3' (SEQ ID NO:17).

[0043] Reverse primer: 5'-ACTGCTGCATAGGCTTTGG-3' (SEQ ID NO:18).

[0044] Nb-PR2b: Forward primer: 5'-AGGTGTTTGCTATGGAATGC-3' (SEQ ID NO:19).

[0045] Reverse primer: 5'-TCTGTACCCACCATCTTGC-3' (SEQ ID NO:20).

[0046] Nb-RbohB: Forward primer: 5'-TCACAAGAGCTCAGGCGTTT-3' (SEQ ID NO:21).

[0047] Reverse primer: 5'-TCATCGAACCGCTTCTCGAC-3' (SEQ ID NO:22).

[0048] Example 8: Detection of H2O2 accumulation in tobacco leaves induced by ThSCSP_20 (1) After ThSCSP_20 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.

[0049] Depend on Figure 4 As shown in Figure b, transient expression of ThSCSP_20 in tobacco leaves induces the accumulation of H2O2.

[0050] Example 9: Detection of Tobacco Callose Deposition Induced by ThSCSP_20 (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_20 respectively; (2) After 24 hours of induction, the treated tobacco leaves were taken out and placed in Callose decolorizing solution, and heated in an oven at 65°C for 1-2 hours, during which time it can be shaken 2-3 times to ensure complete decolorization, and attention should be paid to avoid light; (3) After the chlorophyll of the tobacco leaves was completely decolorized, the decolorizing solution was discarded, and an appropriate volume of 50% ethanol and ddH2O was added for 1-2 times of washing; (4) The tobacco leaves were placed in Callose staining solution for 30-60 minutes of staining; (5) The tobacco leaves were taken out, spread on a glass slide, and an appropriate amount of 50% glycerol was added, and the callose was detected under ultraviolet light. Callose decolorizing solution: 80 mL lactic acid, 80 mL glycerol, 80 mL ddH2O, 80 mL phenol, and 640 mL ethanol; Callose staining solution: 500 mL ddH2O was added with 17.1165 g K2HPO4·3H2O, the pH was adjusted to 9.5, 0.05 g aniline blue was added, and it was stored in the dark.

[0051] As can be seen from the c graph in FIG. 1, Figure 4 As can be seen from the c graph in FIG. 1,

[0052] Example 10: Subcellular localization of ThSCSP_20 The ThSCSP_20-SP sequence was constructed on the PHB-YFP vector by designing specific primers and using T4 ligase. The successfully ligated PHB-ThSCSP_20-SP-YFP vector was transformed into Agrobacterium GV3101, injected into tobacco, and observed by confocal microscopy. The specific steps are as follows: (1) 0.1-0.5 µL DNA and 10-20 µL competent cells (GV3101) were taken into a 1.5 mL EP tube and mixed well; (2) Place on ice for 30 min, and in liquid nitrogen for 5 min; (3) Take out from liquid nitrogen quickly, and place in a 37°C water bath for 5 min; (4) Add 200-400 µL of LB liquid antibiotic-free medium, mix gently, and incubate in a 28°C 200 rpm shaker for 4-5 h until OD600=1.0; (5) Centrifuge (4000 rpm) for 10 min, discard the supernatant, and leave about 200 µL of supernatant to mix gently with a gun head; (6) Spread on the corresponding antibiotic culture dish and incubate in a 28°C incubator for 2 days; (7) Colony PCR identification; (8) Shake the positive clones and extract the plasmid for verification; (9) Tobacco injection, the positive clones transformed by Agrobacterium were sucked into 5 μL to 1 mL of corresponding resistant LB liquid medium, and expanded culture for 2-3 days; (10) 600 μL of bacterial liquid was sucked into 2 mL EP tube, centrifuged (6000 rpm) for 5 min, and the supernatant was discarded; (11) 1.5 mL of pre-prepared MMA Buffer (10 mM MgCl2, 10 mM MES pH=5.8 and 100 μM AS) was added, mixed gently with a gun head, adjusted to OD600=0.7-1.0, and placed in the dark for 3-4 h at room temperature; (12) The bacterial liquid was sucked with a syringe, and the bacterial liquid was gradually pushed into the lower epidermis cells of the leaves, and was marked with a pen or label paper; (13) The injected tobacco plants were cultured in the dark for 12 h, and then placed in a normal incubator, and observed and photographed after 2 days. The primers used are as follows: Forward primer: 5'-accagtctctctctcaagcttATGCTTCCAGCTACCAGCTCTG-3'(SEQ ID NO:23).

[0053] Reverse primer: 5'-gctcaccatactagtggatccTTAAGCCCGGGTAATCTTGTAATG-3'(SEQ ID NO:24).

[0054] The results are shown in Figure 5 The PHB-YFP vector (control) was expressed on the cell nucleus and cell membrane, and the fluorescence localization of the ThSCSP_20-SP and PHB-YFP fusion protein expression was consistent, and was expressed in the cell nucleus and cytoplasm. The results show that ThSCSP_20-SP is located in the cell nucleus and cytoplasm in tobacco.

[0055] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Ustilaginoidea virens effector ThSCSP_20, characterized in that, The nucleotide sequence of the effector ThSCSP_20 is shown as SEQ ID NO:

1.

2. The protein encoded by the U. oryzae effector ThSCSP_20 of claim 1, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO:

2.

3. An expression vector, characterized by, The effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 1.

4. A host cell, characterized in that, The effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 1 or the expression vector according to claim 3.

5. A kit characterized in that, The effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 1 or the protein encoded by the effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 2 or the expression vector according to claim 3 or the host cell according to claim 4.

6. The use of the effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 1 or the protein encoded by the effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 2 in eliciting plant immunity, improving plant resistance to rice grain smut, cultivating transgenic plants resistant to rice grain smut, products for plant resistance to rice grain smut, and designing molecular targets of pesticides.

7. Use according to claim 6, characterized in that, The approach for eliciting plant immunity is at least one selected from the following ①-③: ① inducing the expression of resistance genes; ② inducing the accumulation of H2O2; ③ inducing the deposition of callose.

8. An agent for controlling rice blast, characterized by, The protein encoded by the effector ThSCSP_20 of Ustilaginoidea virens for rice grain smut according to claim 2.

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