Application of sheath blight pathogen effector protein in improving rice disease resistance

Abstract

The present invention relates to the technical field of protein applications, and particularly to the application of Rhizoctonia solani effector proteins in enhancing the disease resistance of rice. The amino acid sequence of the Rhizoctonia solani effector protein is shown as SEQ ID NO.1. The Rhizoctonia solani effector protein provided by the present invention can significantly enhance the disease resistance of rice to Rhizoctonia solani, and significantly up-regulate the expression of rice defense genes, providing a theoretical and practical basis for the research on rice disease resistance.

Description

Technical Field

[0001] The present invention relates to the technical field of protein applications, and specifically relates to the application of Rhizoctonia solani effector proteins in enhancing the disease resistance of rice. Background Art

[0002] Sheath blight of rice is a disease caused by Rhizoctonia solani Rhizoctonia solani ), which seriously affects the high and stable yield of rice. Traditional control methods include agricultural control and chemical control, but these methods have many limitations, such as environmental pollution and the increase of pathogen drug resistance. Therefore, it is particularly important to develop new and environmentally friendly disease resistance strategies.

[0003] In the interaction between plants and pathogens, effector proteins play a key role. Effector proteins are a class of protein molecules that can manipulate the host's innate immune response and enhance the parasitic infection of pathogens in the host. The previous paper published by the inventor, "Cao B, Wang J, Ma J, et al. Large-Scale Screening and Function Analysis of Rhizoctonia solani Effectors Targeting Rice Chloroplasts. [J]. Journal of agricultural and food chemistry, 2024", revealed that the Rhizoctonia solani effector protein AG1IA_08025 can significantly induce plant cell death, but whether this effector protein has other functions, specifically whether the host plant can use this effector protein to counter pathogens is unknown. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an application of Rhizoctonia solani effector proteins in enhancing the disease resistance of rice.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] An application of a Rhizoctonia solani effector protein in enhancing the disease resistance of rice, wherein the amino acid sequence of the Rhizoctonia solani effector protein is as shown in SEQ ID NO.1, the nucleotide sequence of the coding gene of the Rhizoctonia solani effector protein is as shown in SEQ ID NO.2, and the Rhizoctonia solani effector protein can inhibit Rhizoctonia solani of rice and enhance the disease resistance of rice and the expression of defense genes.

[0007] Further, the disease resistance refers to resistance to sheath blight of rice caused by Rhizoctonia solani.

[0008] Further, the Rhizoctonia solani effector protein is used to enhance the expression of defense genes in rice.

[0009] Furthermore, the defense genes include the OsPR10a gene and the OsPAL1 gene.

[0010] Further, in the above application, the effector protein of Rhizoctonia solani is dissolved in water to prepare a protein solution, and rice seeds are soaked with the protein solution, and after the rice seeds are planted, the protein solution is sprayed before rice tillering.

[0011] Further, the rice seeds are soaked with the protein solution until germination.

[0012] Furthermore, the concentration of the protein solution for soaking the rice seeds until germination is 0.01 μM.

[0013] Furthermore, the concentration of the protein solution sprayed before rice tillering is 0.05 μM.

[0014] Compared with the prior art, the present invention has the following beneficial effects.

[0015] 1. The present invention first proves that the effector protein AG1IA_08025 of Rhizoctonia solani can inhibit Rhizoctonia solani, significantly improve the disease resistance of rice to Rhizoctonia solani, and significantly increase the expression of rice defense genes, providing a theoretical and practical basis for the disease resistance research of rice.

[0016] 2. The present invention soaks rice seeds with a protein solution containing the effector protein AG1IA_08025 of Rhizoctonia solani until germination, and then sprays it before rice tillering, achieving a significant effect of inhibiting Rhizoctonia solani and improving the disease resistance of rice to Rhizoctonia solani. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the effect of evaluating the disease resistance of the effector protein of Rhizoctonia solani on Zhonghua 11 by the detached leaf inoculation method in Example 3 of the present invention. A is the apparent diagram of the decay of the detached leaves of Zhonghua 11 including three parallel samples after treatment with the effector protein of Rhizoctonia solani, and B is the statistical chart of the percentage of the decay area of the detached leaves of Zhonghua 11 after treatment with the effector protein of Rhizoctonia solani.

[0018] Figure 2 This is the effect of evaluating the disease resistance of the effector protein of Rhizoctonia solani on Zhonghua 11 by the in vivo leaf sheath inoculation method in Example 3 of the present invention. A is the apparent diagram of the lesion height of Zhonghua 11 including two parallel samples after treatment with the effector protein of Rhizoctonia solani, and B is the statistical chart of the lesion height of Zhonghua 11 after treatment with the effector protein of Rhizoctonia solani.

[0019] Figure 3 This is the expression of defense genes of Beijing 1705 after treatment with the effector protein of Rhizoctonia solani in Example 4 of the present invention. A is the statistical chart of the relative expression level of the OsPR10a gene, and B is the statistical chart of the relative expression level of the OsPAL1 gene. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0021] The Chinese meanings of the English or abbreviations involved in the following embodiments are as follows.

[0022] Kan: Kanamycin.

[0023] Imidazole: Imidazole.

[0024] Tris: Tris(hydroxymethyl)aminomethane;

[0025] Urea: Urea.

[0026] SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0027] HRLH: Highest relative lesion height.

[0028] PR: Defense.

[0029] IPTG: Isopropyl-β-D-thiogalactoside.

[0030] PBS: Phosphate-buffered saline with a mass percentage concentration of 0.7%.

[0031] PDA: Potato dextrose agar.

[0032] RNase: Ribonuclease.

[0033] RNase-Free: RNase-free.

[0034] DNase I: Deoxyribonuclease I.

[0035] RNase-Free Water: Enzyme-free and sterile water.

[0036] DTT: Dithiothreitol.

[0037] PCR: Polymerase chain reaction.

[0038] qRT-PCR: Real-time fluorescence quantitative PCR.

[0039] ROX Reference Dye: Fluorescent quantitative PCR reference dye.

[0040] Example 1.

[0041] This embodiment provides a sheath blight pathogen effector protein. The amino acid sequence of the sheath blight pathogen effector protein is shown in SEQ IN NO.1, and its nucleotide sequence is shown in SEQ ID NO.2, with the gene number being AG1IA_08025. Therefore, the sheath blight pathogen effector protein will also be denoted as AG1IA_08025 hereafter.

[0042] SEQ ID NO.1: MAQLVLAATTAAALAPHAAEVVTLATTVSQAVTIFTKSLKTARDRHHSAQDIGFQTWIRKAEERSRAYYENGPSGPVSWVFTHGTEIPQNALVCGEDIDGNALYVCRTFHRGGVHFGKASRGYKTGAMFGYDGKELEIEFYEVLVADEHTVRWETATYPFIVRKYNGGTLVEGGNEHDGSPLFIARAFYWGGTHPGKTSSMLKGANITFAGKEHCMKDYQVLVLNDPGYASIAALELD.

[0043] SEQ ID NO.2: ATGGCCCAACTTGTTCTCGCTGCGACTACTGCTGCTGCATTGGCACCTCATGCTGCCGAGGTTGTGACTCTTGCAACCACAGTCTCCCAGGCTGTTACCATCTTTACCAAATCCTTAAAGACAGCTAGAGATAGACATCACTCGGCACAGGATATTGGATTCCAGACCTGGATACGAAAGGCAGAGGAAAGATCCAGGGCGTATTACGAAAACGGACCGAGTGGTCCCGTTTCATGGGTGTTTACGCACGGGACGGAAATTCCCCAAAATGCGCTTGTGTGTGGGGAAGATATTGATGGGAATGCGTTGTACGTGTGTAGGACGTTCCATAGGGGAGGAGTCCACTTTGGAAAGGCCAGCCGGGGATACAAGACTGGTGCCATGTTTGGATACGATGGCAAAGAACTCGAGATCGAGTTTTACGAAGTCTTAGTAGCTGACGAACACACCGTCCGATGGGAAACTGCGACATACCCATTTATCGTTCGAAAGTACAACGGGGGAACATTAGTAGAGGGCGGAAACGAACACGACGGATCACCGTTATTCATTGCCAGAGCCTTTTACTGGGGCGGAACTCATCCCGGAAAGACCAGCTCGATGCTCAAGGGTGCTAATATCACCTTTGCAGGGAAAGAACATTGTATGAAAGATTACCAGGTACTTGTTCTGAACGATCCCGGATACGCGAGTATAGCAGCACTGGAGCTGGATTGA。

[0044] Example 2.

[0045] This example provides the preparation of the effector protein of Rhizoctonia solani.

[0046] 1. Strain culture and induced expression.

[0047] (1) Strain culture and induced expression.

[0048] Construction of recombinant plasmid: The gene shown in SEQ ID NO.2 was ligated between the BamH I and Sal I sites of the pET28a vector to obtain a recombinant plasmid.

[0049] Strain culture: The constructed recombinant plasmid was transformed into the Escherichia coli expression strain BL21(DE3). Single colonies were picked and inoculated into LB liquid medium containing the antibiotic Kan, and cultured overnight at 37°C.

[0050] Induced expression: The overnight cultured bacterial solution was inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C and 200 rpm until the OD 600 reached 0.6. IPTG was added to a final concentration of 0.5 mM, and the induction temperature was adjusted according to the protein characteristics. In this example, induction was carried out at 28°C for 8 h.

[0051] 2. Bacterial cell collection.

[0052] The induced bacterial solution was cooled at 4°C for 10 minutes, then centrifuged at 4°C and 6000 r / min for 10 minutes to collect the bacterial cells, and the supernatant was discarded.

[0053] 3. Cell disruption.

[0054] Resuspension: The bacterial cells were resuspended with PBS buffer.

[0055] Disruption: An ultrasonic cell disruptor was used for disruption until the bacterial cells were completely disrupted. The disruption conditions were a power of 600 W, disruption for 3 seconds, and pause for 5 seconds. The disruption process was carried out in an ice bath to prevent protein denaturation.

[0056] 4. Crude protein extraction.

[0057] The disrupted bacterial solution was centrifuged at 4°C and 12000 g for 20 minutes to separate the supernatant and the precipitate. The supernatant contains soluble proteins, and the precipitate may contain inclusion body proteins.

[0058] 5. Protein purification.

[0059] (1) 500 μL of Ni-NTA suspension was added to the purification column. After washing the column bed 5 times with 5 mL of Buffer B, the soluble protein extract shown in Table 1 was added.

[0060] Table 1: Formulation of soluble protein extract

[0061]

[0062] (2) When the protein extract flowed to below the upper edge of the column bed, 5 mL of Buffer B was added to wash the column twice.

[0063] (3) When Buffer B flowed to below the upper edge of the column bed, 5 mL of Buffer C shown in Table 2 was added to wash the column twice.

[0064] Table 2: Formulation of Buffer C

[0065]

[0066] (4) When the Buffer C in the column bed has drained completely, add 1 mL of Buffer E as shown in the formulation of Table 3 to elute the target protein.

[0067] Table 3: Formulation of Buffer E

[0068]

[0069] (5) Add the eluted target protein solution into a dialysis bag and dialyze it in 1×PBS solution for 48 h. Replace the dialysis solution every 3 h during this period.

[0070] (6) After dialysis, measure the protein concentration using a BCA kit, detect the protein purity by SDS-PAGE, and store it at -20 °C.

[0071] Example 3.

[0072] This example provides the application of the effector protein of Rhizoctonia solani in improving the disease resistance of rice.

[0073] 1. Test method.

[0074] The Rhizoctonia solani used in this example is Rhizoctonia solani AG1-IA, and the tested rice variety is Zhonghua 11.

[0075] In the experimental group, rice seeds were soaked in a solution of the effector protein of Rhizoctonia solani at a concentration of 0.01 μM. After germination, they were planted in the conventional way. Before rice tillering, a solution of the effector protein of Rhizoctonia solani at a concentration of 0.05 μM was sprayed. 24 h later, Rhizoctonia solani was inoculated on the rice and samples were taken for detection of disease resistance indexes. Treatment with sterile water was used as the control group.

[0076] The inoculation methods include the detached leaf inoculation method and the living leaf sheath inoculation method.

[0077] Detached leaf inoculation method.

[0078] Cut a 4-cm-long part from the middle of the leaf and lay it flat in a petri dish containing filter paper for moisture retention, with the front side of the leaf closely attached to the surface of the culture medium. At the same radius of the petri dish containing Rhizoctonia solani, use a surface-sterilized punch to take a 7-mm-diameter PDA block with mycelium, and place the PDA block in the middle part of the detached leaf, with the side with mycelium closely attached to the abaxial surface of the leaf. Seal it with a sealing film and incubate it at 28 °C. Each petri dish is used as 1 inoculation replicate. Among them, 3 detached leaf tissues are taken from each rice variety, and 1 agar block with bacteria is inoculated on each leaf. Observe the apparent rot of the leaf 3 days after inoculation and calculate the percentage of the rotten area in the leaf.

[0079] In vivo leaf sheath inoculation method.

[0080] Place a mycelial cake containing Rhizoctonia solani of rice into the center of a PDA medium plate. Place a circle of sterile dry bark with a length of 1 cm and a width of 5 mm around the culture dish. After sealing the plate, incubate it upright at 28 °C for 3 days. At the early tillering stage of rice planted in small pots, select the first leaf sheath separated from the main stem. Use forceps to pick up a thin piece of bark with Rhizoctonia solani mycelia covering the surface, place the side with mycelia facing the leaf sheath at the base of the rice stem, and wrap the inoculated part of the bark with plastic wrap. After inoculation, transfer the seedlings to a shaded place on the west balcony and spray water to keep the humidity twice a day, once in the morning and once in the evening. After 10 days of inoculation, cut the plants from the base of the rice stem and measure the lesion height based on the cut part and the height of the whole rice plant with a ruler, and calculate the HRLH to evaluate the disease incidence.

[0081] 2. Test results.

[0082] The results are as Figure 1 and Figure 2 shown. The disease resistance was evaluated by using the detached leaf inoculation method and the in vivo leaf sheath inoculation method. It was found that after treatment with the protein solution, the diseased area of the detached leaves and the highest relative lesion height of the in vivo rice were significantly reduced compared with the control group, p < 0.05, indicating that the pathogenic ability of Rhizoctonia solani on rice leaves was significantly reduced and the disease resistance of rice was significantly improved.

[0083] Example 4.

[0084] This example provides the application of the effector protein of Rhizoctonia solani in increasing the expression level of rice defense genes.

[0085] 1. Test method.

[0086] Using Beijingjing 1705 as the test variety, in the experimental group, rice seeds were soaked in a solution of the effector protein of Rhizoctonia solani with a concentration of 0.01 μM. After germination, they were planted in the conventional way. Before rice tillering, a solution of the effector protein of Rhizoctonia solani with a concentration of 0.05 μM was sprayed. After 24 hours of spraying, the expression levels of the OsPR10a gene with gene ID Os12g0555500 and the OsPAL1 gene with gene ID Os02g0627100 were detected, and the treatment with sterile water was used as the control group. The specific process is as follows.

[0087] 1.1 Extraction of total RNA.

[0088] The extraction of total plant RNA was carried out with reference to the instruction manual of the Ultra-pure RNA Extraction Kit with the product number CW0597 produced by ComWin Biotech Co., Ltd. The specific operation is as follows.

[0089] (1)Sample treatment

[0090] 1) Tissue homogenization: After thoroughly grinding 30 mg of tissue in liquid nitrogen, add 1 mL of Buffer RLT.

[0091] 2) Monolayer cultured cells: Discard the culture medium, add an appropriate amount of Buffer RLT. For every 10 cm 2 Add 1 mL of Buffer RLT.

[0092] 3) Cell suspension: Centrifuge to collect cells. For every 5×10 6 cells, add 1 mL of Buffer RLT.

[0093] (2) After adding Buffer RLT to the sample, pipette up and down several times to fully lyse the sample. Let it stand at room temperature for 5 min to completely separate the protein-nucleic acid complex.

[0094] (3) Add chloroform at a ratio of 200 μL of chloroform per 1 mL of Buffer RLT, cover the tube cap, shake vigorously for 15 seconds, and let it stand at room temperature for 2 min.

[0095] (4) Centrifuge at 12,000 rpm for 10 min at 4 °C. At this time, the sample is divided into three layers: a red organic phase, a middle layer, and an upper colorless aqueous phase. RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube.

[0096] (5) Add an equal volume of 70% ethanol solution to the obtained aqueous solution and invert to mix well.

[0097] (6) Add all the solution obtained in the previous step to the adsorption column placed in a 2 mL collection tube. Centrifuge at 12,000 rpm for 20 seconds, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0098] (7) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 20 seconds, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0099] (8) Prepare the DNase I mixture: Take 52 μL of RNase-Free Water, add 8 μL of 10× Reaction Buffer and 20 μL of DNase I with a concentration of 1 U / μL, mix well to prepare a DNase I mixture with a final volume of 80 μL.

[0100] (9) Directly add 80 μL of the DNase I mixture to the adsorption column and incubate at 280 °C for 15 min.

[0101] (10) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and place the adsorption column back into the collection tube;

[0102] (11) Add 500 μL of Buffer RW2 to the adsorption column, centrifuge at 12,000 rpm for 20 s, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0103] (12) Repeat step (11).

[0104] (13) Centrifuge at 12,000 rpm for 2 min, pour out the waste liquid in the collection tube. Place the adsorption column at room temperature for several minutes to dry completely.

[0105] (14) Place the adsorption column into a new RNase-Free centrifuge tube, add 40 μL of RNase-Free Water to the middle part of the adsorption column, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70 °C to prevent degradation.

[0106] 1.2 Reverse transcription.

[0107] The reverse transcription system of rice RNA refers to the instruction manual of M-MLV reverse transcriptase from Invitrogen. The steps are as follows.

[0108] (1) Add 1 μL of Oligo dT with a concentration of 100 μM, 4 μL of dNTP with a concentration of 2.5 mM, and 2 μg of total RNA to a nuclease-free microcentrifuge tube, and supplement with RNase-Free water to 12 μL.

[0109] (2) After heating the mixture at 65 °C for 5 min, quickly place it on ice for 2 min. After brief centrifugation, add 4 μL of 5× First Strand Synthesis Buffer, 2 μL of DTT with a concentration of 0.1 M, and 1 μL of RNase inhibitor.

[0110] (3) Gently mix all components and incubate at 37 °C for 2 min.

[0111] (4) Add 1 μL of M-MLV reverse transcriptase at room temperature, gently pipette and mix well, and incubate at 37 °C for 50 min.

[0112] (5) Heat at 70 °C for 15 min to terminate the reaction.

[0113] 1.3 Fluorescent quantitative PCR to detect the expression of PR gene in transgenic rice.

[0114] Use SYBR ® Premix Ex TaqTM Detection was performed using a fluorescence quantitative PCR kit, and the reaction system is shown in Table 4 below.

[0115] Table 4: qRT-PCR reaction system

[0116]

[0117] Reaction procedure: Pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, extension at 60°C for 31 s, for a total of 40 cycles.

[0118] The 2 -ΔΔCt method was used to calculate gene expression, and the relative expression level of the target gene = 2 -ΔΔCt , where -ΔΔCt = -(ΔCt,a - ΔCt,b), where Ct: fluorescence threshold; a: target gene; b: internal reference gene.

[0119] In this example, the OsActin1 gene with gene ID Os03g0718100 was used as the internal reference gene, and the primer information for OsActin1, OsPR10a, and OsPAL1 for qRT-PCR is shown in Table 5.

[0120] Table 5: Primer information

[0121]

[0122] 2. Test results.

[0123] The results are as Figure 3 shown. Compared with the control group, after spraying the effector protein for 24 h, the expression of rice PR genes OsPR10a and OsPAL1 in the experimental group was significantly induced and increased, p < 0.05, and the increase multiples reached 50 to 90 times. The increase in PR genes is an active response of plants to biotic or abiotic stresses, aiming to enhance their survival ability, and this process is crucial for the health and environmental adaptation of plants. The results of the present invention lay a foundation for studying the mechanism of rice's response to bacterial diseases and provide a new option for controlling rice bacterial diseases.

[0124] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. Application of effector protein of sheath blight fungus in improving disease resistance of rice, characterized in that: The amino acid sequence of the sheath blight effector protein is shown in SEQ ID NO.1, and the disease resistance refers to resistance to rice sheath blight caused by Rhizoctonia solani.

2. The use of the sheath blight effector protein according to claim 1 in improving rice disease resistance, characterized in that: The sheath blight pathogen effector protein is used to increase the expression of rice defense genes, and the defense genes are OsPR10a gene and OsPAL1 gene.

3. The use of the sheath blight effector protein according to claim 1 in improving rice disease resistance, characterized in that: In the application, the sheath blight pathogen effector protein is dissolved in water to prepare a protein solution, rice seeds are soaked in the protein solution, and after the rice seeds are planted, the protein solution is sprayed before the rice tillers.

4. The use of the sheath blight effector protein in improving rice disease resistance according to claim 3, characterized in that: The rice seeds are soaked in the protein solution until they germinate.

5. The use of the sheath blight effector protein according to claim 4 in improving rice disease resistance, characterized in that: The concentration of the protein solution used to soak rice seeds until germination was 0.01 μM.

6. The use of the sheath blight effector protein in improving rice disease resistance according to claim 5, characterized in that: The concentration of the protein solution sprayed before rice tillering is 0.05 μM.

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