Application of rhizoctonia solani effect protein in improving disease resistance of rice

Abstract

The invention relates to the technical field of protein application, in particular to application of rhizoctonia solani effect protein to improvement of rice disease resistance. The amino acid sequence of the rhizoctonia solani effect protein is as shown in SEQ ID NO. 1. The rhizoctonia solani effect protein provided by the invention can obviously improve the disease resistance of rice to the rice sheath blight disease and obviously improve the expression of a rice defensive gene, and the result provides a theoretical and practical basis for the research on the disease resistance of the rice.

Description

Technical Field

[0001] The invention relates to the technical field of protein application, and in particular to the application of sheath blight pathogen effector protein in improving rice disease resistance. Background Art

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

[0003] Effector proteins play a key role in the interaction between plants and pathogens. 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 inventor's previously published paper "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 effector protein AG1IA_08025 of Rhizoctonia solani can significantly induce plant cell death, but whether the effector protein has other effects, such as whether the host plant can use the effector protein to counter pathogens, is still unknown. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of an effector protein of sheath blight fungus in improving the disease resistance of rice.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A use of a sheath blight pathogen effector protein in improving rice disease resistance, wherein the amino acid sequence of the sheath blight pathogen effector protein is shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the sheath blight pathogen effector protein is shown in SEQ ID NO.2, and the sheath blight pathogen effector protein can inhibit rice sheath blight pathogen and improve rice disease resistance and defense gene expression.

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

[0008] Furthermore, the sheath blight pathogen effector protein is used to increase the expression of rice defense genes.

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

[0010] Furthermore, in the application, the sheath blight fungus 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.

[0011] Furthermore, the rice seeds are soaked in the protein solution until they germinate.

[0012] Furthermore, the concentration of the protein solution used to soak the rice seeds until they germinate 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 confirms for the first time that the effector protein AG1IA_08025 of sheath blight fungus can inhibit rice sheath blight fungus, significantly improve the disease resistance of rice to rice sheath blight, and significantly increase the expression of rice defense genes, providing a theoretical and practical basis for rice disease resistance research.

[0016] 2. The present invention soaks rice seeds in a protein solution containing the effector protein AG1IA_08025 of rice sheath blight until they germinate, and then sprays the solution before the rice tillers, thereby achieving the effect of significantly inhibiting rice sheath blight and improving the disease resistance of rice to rice sheath blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of the effector protein of Rhizoctonia solani on the disease resistance of Zhonghua 11 was evaluated by the in vitro leaf inoculation method in Example 3 of the present invention. A is an apparent image of the rot of the in vitro leaves of Zhonghua 11 including three parallel samples after treatment with the effector protein of Rhizoctonia solani, and B is a statistical graph of the percentage of rot area of ​​the in vitro leaves of Zhonghua 11 after treatment with the effector protein of Rhizoctonia solani.

[0018] Figure 2 The effect of the effector protein of Rhizoctonia solani on the disease resistance of Zhonghua 11 was evaluated by the living leaf sheath inoculation method in Example 3 of the present invention. A is an apparent graph of the lesion height of Zhonghua 11 including two parallel samples after treatment with the effector protein of Rhizoctonia solani, and B is a statistical graph of the lesion height of Zhonghua 11 after treatment with the effector protein of Rhizoctonia solani.

[0019] Figure 3 1 is the expression of defense genes in Beijing 1705 after treatment with the effector protein of Rhizoctonia solani in Example 4 of the present invention, A is a statistical diagram of the relative expression of the OsPR10a gene, and B is a statistical diagram of the relative expression of the OsPAL1 gene. DETAILED DESCRIPTION

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

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

[0022] Kan: kanamycin.

[0023] Imidazole: imidazole.

[0024] Tris: tris(hydroxymethyl)aminomethane; Urea: urea.

[0025] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0026] HRLH: highest relative lesion height.

[0027] PR: Defense.

[0028] IPTG: isopropyl-β-D-thiogalactoside.

[0029] PBS: Phosphate-balanced saline with a mass percentage concentration of 0.7%.

[0030] PDA: potato dextrose agar.

[0031] RNase: ribonuclease.

[0032] RNase-Free: RNase-free.

[0033] DNase I: deoxyribonuclease I.

[0034] RNase-Free Water: Enzyme-free sterile water.

[0035] DTT: dithiothreitol.

[0036] PCR: polymerase chain reaction.

[0037] qRT-PCR: real-time quantitative PCR.

[0038] ROX Reference Dye: Fluorescent quantitative PCR reference dye.

[0039] Example 1.

[0040] The present 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, the nucleotide sequence is shown in SEQ ID NO.2, and the gene number is AG1IA_08025. Therefore, the sheath blight pathogen effector protein will be hereinafter referred to as AG1IA_08025.

[0041] SEQ ID NO.1: MAQLVLAATTAAALAPHAAEVVTLATTVSQAVTIFTKSLKTARDRHHSAQDIGFQTWIRKAEERSRAYYENGPSGPVSWVFTHGTEIPQNALVCGEDIDGNALYVCRTFHRGGVHFG KASRGYKTGAMFGYDGKELEIEFYEVLVADEHTVRWETATYPFIVRKYNGGTLVEGGNEHDGSPLFIARAFYWGGTHPGKTSSMLKGANITFAGKEHCMKDYQVLVLNDPGYASIAALELD.

[0042] SEQ ID NO.2:.

[0043] Example 2.

[0044] This example provides the preparation of effector protein of Rhizoctonia solani.

[0045] 1. Strain culture and induced expression.

[0046] (1) Strain culture and induced expression.

[0047] Recombinant plasmid construction: The gene shown in SEQ ID NO.2 was connected to the pET28a vector Bm I and Sal I site to obtain a recombinant plasmid.

[0048] Strain culture: The constructed recombinant plasmid was transformed into the E. coli expression strain BL21 (DE3). A single clone was selected and inoculated into LB liquid medium containing the antibiotic Kan and cultured at 37°C overnight.

[0049] Induce expression: inoculate the overnight culture into fresh LB medium at a ratio of 1:100, and culture at 37°C and 200 rpm until OD 600 IPTG was added to a final concentration of 0.5 mM, and the induction temperature was adjusted according to the protein characteristics. In this embodiment, the induction was performed at 28° C. for 8 h.

[0050] 2. Bacteria collection.

[0051] The induced bacterial solution was cooled at 4°C for 10 minutes, and then centrifuged at 4°C and 6000 r / min for 10 minutes to collect the bacteria and discard the supernatant.

[0052] 3. Cell disruption.

[0053] Resuspend: Resuspend the cells in PBS buffer.

[0054] Disruption: Use an ultrasonic cell disruptor to disrupt the cells until they are completely broken. The conditions are 600W, 3 seconds of disruption, and 5 seconds of pause. The disruption process is carried out in an ice bath to prevent protein denaturation.

[0055] 4. Extraction of crude protein.

[0056] The disrupted bacterial solution was centrifuged at 4°C and 12,000 g for 20 minutes to separate the supernatant and the precipitate. The supernatant contains soluble proteins, and the precipitate may contain inclusion body proteins.

[0057] 5. Protein purification.

[0058] (1) Add 500 μL of Ni-NTA suspension to the purification column, rinse the column bed five times with 5 mL of Buffer B, and then add the soluble protein extract shown in Table 1.

[0059] Table 1: Soluble protein extract formula

[0060] (2) When the protein extract flows below the top of the column bed, add 5 mL of Buffer B to wash the column twice.

[0061] (3) When Buffer B flows below the top of the column bed, add 5 mL of Buffer C (see Table 2) to wash the column twice.

[0062] Table 2: Buffer C formula

[0063] (4) When Buffer C in the column bed has run out, add 1 mL of Buffer E as shown in Table 3 to elute the target protein.

[0064] Table 3: Buffer E formula

[0065] (5) Add the eluted target protein solution into the dialysis bag and dialyze it in 1× PBS solution for 48 hours. The dialysate was replaced every 3 hours.

[0066] (6) After dialysis, the protein concentration was determined by BCA kit and the protein purity was detected by SDS-PAGE. The protein was stored at -20°C.

[0067] Example 3.

[0068] This embodiment provides the use of sheath blight fungus effector protein in improving rice disease resistance.

[0069] 1. Test method.

[0070] The rice sheath blight pathogen used in this example is Rhizoctonia solani AG1-IA, and the rice variety tested is Zhonghua 11.

[0071] The experimental group was to soak rice seeds in a 0.01μM solution of sheath blight effector protein, plant them in the conventional way after germination, spray them with a 0.05μM solution of sheath blight effector protein before tillering, inoculate rice sheath blight 24 hours later and take samples for disease resistance index detection. Sterile water treatment was used as the control group.

[0072] Inoculation methods include in vitro leaf inoculation and in vivo leaf sheath inoculation.

[0073] In vitro leaf inoculation method.

[0074] Cut a 4 cm long section from the middle of the leaf and spread it flat in a culture dish containing filter paper for moisture retention, with the front of the leaf close to the surface of the culture medium. At the same radius of the culture dish containing rice sheath blight, use a surface-sterilized puncher to take a 7 mm diameter PDA block with hyphae, and place the PDA block in the middle of the detached leaf, with the side with hyphae close to the abaxial surface of the leaf. Seal with sealing film and culture at 28°C. Each culture dish is used as an inoculation replicate, in which 3 detached leaf tissues are taken from each rice variety, and each leaf is inoculated with 1 agar block with bacteria. Observe the appearance of leaf rot 3 days after inoculation, and calculate the percentage of rotten area to leaf.

[0075] Living leaf sheath inoculation method.

[0076] Place the fungus cake containing rice sheath blight in the center of the PDA medium plate, place a circle of sterile dry bark 1 cm long and 5 mm wide around the culture dish, seal the plate and culture it at 28℃ for 3 days. In the early stage of tillering of rice planted in small pots, select the first leaf sheath separated from the main stem. Use tweezers to grab a piece of thin bark covered with hyphae of sheath blight, with the hyphae facing the leaf sheath, place it at the base of the rice stem and wrap the inoculated part of the bark with plastic wrap. The inoculated seedlings are transferred to the shade of the west balcony and sprayed with water once in the morning and evening every day to keep them moist. 10 days after inoculation, cut the plants from the base of the rice stem and measure the height of the lesions and the height of the whole rice plant with a ruler based on the cut part as the baseline, and calculate HRLH to evaluate the disease situation.

[0077] 2. Test results.

[0078] The results are as follows Figure 1 and Figure 2 As shown, the disease resistance was evaluated by detached leaf inoculation method and living leaf sheath inoculation method. It was found that after protein solution treatment, the disease area of ​​detached leaves and the highest relative lesion height of living rice were significantly reduced compared with the control group, p<0.05, indicating that the disease ability of rice sheath blight fungus on rice leaves was significantly reduced and the disease resistance of rice was significantly improved.

[0079] Example 4.

[0080] This example provides the use of sheath blight fungus effector proteins in increasing the expression level of rice defense genes.

[0081] 1. Test method.

[0082] Beijing 1705 was used as the test variety. The experimental group was to soak the rice seeds in a 0.01 μM solution of sheath blight effector protein. After germination, the rice seeds were planted in a conventional manner. The sheath blight effector protein solution with a concentration of 0.05 μM was sprayed before the rice tillers. After 24 hours of spraying, the expression levels of the OsPR10a gene with a gene ID of Os12g0555500 and the OsPAL1 gene with a gene ID of Os02g0627100 were detected. Sterile water treatment was used as the control group. The specific process is as follows.

[0083] 1.1 Extraction of total RNA.

[0084] The extraction of plant total RNA refers to the instructions of the ultra-pure RNA extraction kit with product number CW0597 produced by Kangwei Century Biological Company. The specific operation is as follows.

[0085] (1) Sample processing 1) Tissue homogenate: Grind 30 mg of tissue thoroughly in liquid nitrogen and add 1 mL of Buffer RLT.

[0086] 2) Monolayer cell culture: discard the culture medium and add an appropriate amount of Buffer RLT per 10cm 2 Add 1 mL of Buffer RLT.

[0087] 3) Cell suspension: Collect cells by centrifugation. 6 Add 1 mL of Buffer RLT per 1 cell.

[0088] (2) After adding Buffer RLT to the sample, pipette and vortex several times to fully lyse the sample. Leave at room temperature for 5 minutes to completely separate the protein-nucleic acid complex.

[0089] (3) Add chloroform at a ratio of 200 μL chloroform per 1 mL of Buffer RLT, cap the tube, shake vigorously for 15 seconds, and leave at room temperature for 2 minutes.

[0090] (4) Centrifuge at 4°C, 12,000 rpm for 10 min. The sample will then separate into three layers: a red organic phase, a middle layer, and an upper colorless aqueous phase. The RNA is mainly in the upper aqueous phase. Transfer the upper aqueous phase to a new RNase-Free centrifuge tube.

[0091] (5) Add an equal volume of 70% ethanol solution to the aqueous solution and mix thoroughly by inversion.

[0092] (6) Add all the solution obtained in the previous step to the adsorption column in the 2 mL collection tube. Centrifuge at 12000 rpm for 20 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0093] (7) Add 350 μL of Buffer RW1 to the adsorption column and centrifuge at 12,000 rpm for 20 seconds. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0094] (8) Prepare DNase I mixed solution: Take 52 μL of RNase-Free Water, add 8 μL of 10× Reaction Buffer and 20 μL of 1 U / μL DNase I, mix well, and prepare a final volume of 80 μL of DNase I mixed solution.

[0095] (9) Add 80 μL of DNase I mixture directly to the adsorption column and incubate at 280°C for 15 min.

[0096] (10) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 10,000 rpm for 1 min, discard the waste liquid, and return the adsorption column to the collection tube; (11) Add 500 μL of Buffer RW2 to the adsorption column and centrifuge at 12,000 rpm for 20 seconds. Discard the waste liquid in the collection tube and place the adsorption column back into the collection tube.

[0097] (12) Repeat step (11).

[0098] (13) Centrifuge at 12,000 rpm for 2 min and discard the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.

[0099] (14) Place the adsorption column in a new RNase-Free centrifuge tube, add 40 μL of RNase-Free Water to the middle part of the adsorption column, leave it at room temperature for 1 min, centrifuge it at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70°C to prevent degradation.

[0100] 1.2 Reverse transcription.

[0101] The reverse transcriptase system of rice RNA was prepared according to the instruction manual of M-MLV reverse transcriptase from Invitrogen Company, and the steps are as follows.

[0102] (1) Add 1 μL of 100 μM Oligo dT, 4 μL of 2.5 mM dNTP, and 2 μg of total RNA to a nuclease-free microcentrifuge tube and add RNase-Free water to make up to 12 μL.

[0103] (2) The mixture was heated at 65°C for 5 min and then quickly cooled on ice for 2 min. After a brief centrifugation, 4 μL of 5× first-strand synthesis buffer, 2 μL of 0.1 M DTT, and 1 μL of RNase inhibitor were added.

[0104] (3) Gently mix all ingredients and incubate at 37°C for 2 min.

[0105] (4) Add 1 μL of M-MLV reverse transcriptase at room temperature, gently pipette to mix, and incubate at 37°C for 50 min.

[0106] (5) Heat at 70°C for 15 min to terminate the reaction.

[0107] 1.3 Fluorescence quantitative PCR detection of transgenic rice PR gene expression.

[0108] SYBR ® Premix Ex Taq TM The fluorescence quantitative PCR kit was used for detection, and the reaction system was shown in Table 4 below.

[0109] Table 4: qRT-PCR reaction system

[0110] The reaction program was as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and extension at 60°C for 31 s, for a total of 40 cycles.

[0111] Adoption 2 -ΔΔCt Method: Gene expression was calculated, relative expression of target gene = 2 -ΔΔCt , where -ΔΔCt=-(ΔCt, a-ΔCt, b), Ct: fluorescence threshold; a: target gene; b: internal reference gene.

[0112] In this example, the OsActin1 gene with a gene ID of Os03g0718100 was used as an internal reference gene. The primer information of OsActin1, OsPR10a and OsPAL1 used for qRT-PCR is shown in Table 5.

[0113] Table 5: Primer information

[0114] 2. Test results.

[0115] The results are as follows Figure 3 As shown, compared with the control group, the expression of rice PR genes OsPR10a and OsPAL1 was significantly induced and increased in the experimental group 24 hours after spraying the effector protein, p<0.05, and the increase was 50 to 90 times. The increase of PR genes is an active response of plants to biological or abiotic stresses, aiming to enhance their survival ability. This process is crucial to the health and adaptation of plants to the environment. The results of the present invention lay the foundation for studying the mechanism of rice response to bacterial diseases and provide a new option for preventing and controlling rice bacterial diseases.

[0116] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall 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 pathogen effector protein is shown in SEQ ID NO.

1.

2. The use of the sheath blight effector protein according to claim 1 in improving rice disease resistance, characterized in that: The disease resistance refers to resistance to rice sheath blight caused by Rhizoctonia solani.

3. 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.

4. The use of the sheath blight effector protein in improving rice disease resistance according to claim 3, characterized in that: The defense genes include OsPR10a gene and OsPAL1 gene.

5. The use of the sheath blight effector protein in improving rice disease resistance according to claim 1, 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.

6. The use of the sheath blight effector protein in improving rice disease resistance according to claim 5, characterized in that: The rice seeds are soaked in the protein solution until they germinate.

7. The use of the sheath blight effector protein in improving rice disease resistance according to claim 6, characterized in that: The concentration of the protein solution used to soak rice seeds until germination was 0.01 μM.

8. The use of the sheath blight effector protein according to claim 7 in improving rice disease resistance, characterized in that: The concentration of the protein solution sprayed before rice tillering is 0.05 μM.

Images