Preparation of LB Medium

1. Cleaning the conical flask.
2. Weigh LB broth powder at a ratio of 50 mL: 1.25 g and then pouring into the conical flask.
3. Adding ultra-pure water, and seal with a sealing film.
4. Put the conical flask into the high-temperature sterilization pot and set the sterilization parameter as follows: 121℃, 20 min.

(ps: If a solid medium is to be prepared, add 1% AGAR for addition.)

Expanding Cultivation of E. coli

1. Put the pipette, pipette tip, liquid LB medium, marker, and other items into the ultra-clean Bench for UV sterilization for 20 min.
2. Turn off the UV lamp, and turn on the fan and the light. Spray both hands with alcohol and step into the Clean Bench for further operation.
3. Add antibiotics into the LB medium at a ratio of 1:1000 and mix well.
4. Pour 20 mL LB into a 50 mL tube, transfer 200 μL bacterial solution into a 50 mL tube.
5. Place in a 37℃ shaker and incubate overnight.

Plasmid Extraction

1. Check whether RNAase A is added to Buffer P1, whether anhydrous ethanol is added to Wash Solution, and whether Buffer P2 and P3 are precipitating.
2. Take 6 mL of the overnight cultured bacterial solution to the centrifugal tube, centrifuge 8000 ×g for 2 min, collect the bacterial solution and discard the medium.
3. Add 250 μL Buffer P1 to the precipitation and suspend the bacteria.
4. Add 250 µl Buffer P2 and immediately reverse the centrifugal tube for 5-10 mixing times. Set it for 2 min at room temperature.
5. Add 350 µL Buffer P3 and immediately reverse the centrifuge tube for 5-10 times of mixing.
6. Centrifuge 12000 ×g for 10 min. Transfer the supernatant into the adsorption column, centrifuge 8000 ×g for 30 s, and drain the liquid from the collection tube.
7. Add 500 µl Buffer DW1, centrifuge 9000 ×g for 30 s, drain the liquid from the collection tube.
8. Add 500 µl Wash Solution, centrifuge 9000 ×g for 30 s, and drain the liquid from the collection tube.
9. Repeat Step 8.
10. Centrifuge the empty adsorption column at 9000 ×g for 1 min.
11. Place the adsorption column in a clean 1.5 mL centrifuge tube, add 30 µl Elution Buffer in the center of the adsorption column, leave for 1 min at room temperature, and centrifuge for 1 min. Preserve the DNA solution in the tube.

Colonal PCR

35 cycles

Bacterial Liquid PCR

35 cycles

Electrophoresis

1. Prepare agarose powder, glass plate, comb, and TAE solution in advance.
2. Weigh 0.75 g agarose powder and add to the prepared 50 mL 1xTAE solution, mix well, and heat in the microwave oven until the solution is clarified, add red nucleic acid dye, and shake well.
3. Pour into the glass plate, insert the comb, and wait for 10 min until the gel is solidified.
4. Put the solidified glue into the electrophoresis tank, point the sample, and point the marker.
5. Close the cover of the electrophoresis tank, set the voltage to 180 V for 15 min, and start electrophoresis.

Gel Extraction

1. Check whether anhydrous ethanol is added to the Wash Solution and Buffer B2 is precipitated. Adjust the water bath to 50℃.
2. Cut the gel block which containing the target fragment from the agarose gel and weigh it.
3. Add 400 µl Buffer B2 and the gel to the centrifugal tube and melt it to liquid sol in a 50℃ thermostat water bath for 10 min.
4. Transfer the liquid sol into the adsorption column, centrifuge 8000 ×g for 30 s, and drain the liquid in the collection tube.
5. Add 500 µL Wash Solution, centrifuge 9000 ×g for 30 s, and drain the liquid into the collection tube.
6. Repeat Step 5.
7. Centrifuge the empty adsorption column 9000 ×g for 1 min.
8. Put the adsorption column into a clean 1.5 mL centrifuge tube, add the Elution Buffer into the adsorption column，and then set it at room temperature for 1 min. Centrifuge for 1 min. Preserve the DNA solution in the tube.

DNA Digestion with Restriction Endonucleases

1. Prepare 10 µl of Buffer and 1.5 µl of BamH1 and EcoR1 for each, 66 µL of ultrapure water, 12 µl of template of gel extraction material, 9 µl of plasmid extraction material, and two 200 µL tubes in advance.
2. Gel extraction material: Add 32 µl of ultrapure water, 5 µL of Buffer, and 12 µL of Template and Enzyme (BamH1 and EcoR1 0.5 µl each).
3. Plasmid extraction material: Add 34 µl ultrapure water, 5 µL Buffer, 9 µL Template, Enzyme (BamH1 and EcoR1, 1 µl each).
4. Place in a 37C water bath for 1 hour.
5. Point the template and then electrophoresis to confirm whether the strip sizes are correct. If it’s correctly cut off, the gel extraction is carried out.
6. Test the concentration of recycled material.

Verification of Enzyme Cutting

Enzyme cutting at 37℃ for 30 min

Ligation with T4 DNA Ligase

Incubate at 22℃ for 1 h

Transformation of E. coli

1. Add the product to 50 µL of competent cells and mix it well by flicking the bottom of the centrifuge tube.
2. Place the tube on ice for 30 min.
3. 42℃ heat shock for 90 s, immediately put on ice for 2 min.
4. Add 500 µL of non-resistant liquid LB medium into the centrifuge tube into the super-clean bench and incubate it in a 37℃ shaker for 1 hour.
5. Centrifuge 5000 ×g for 1 min.
6. Discard the supernatant, leaving about 100 µL, use a pipette to resuspend the bacteria solution, pour it on the solid medium, and apply it evenly with a coating stick. Wait for the board to dry on a super-clean table, seal it, and put it into a 37℃ incubator for overnight cultivation.

Prokaryotic Expression of DsRNA

1. Transfer the bacteria to a 50 mL tube and shake it overnight.
2. On the second day, transfer 2 mL of overnight cultured bacterial solution to 100 mL LB medium in a ratio of 1: 50, and cultured in a 37℃ shaking table for 4 h.
3. IPTG with 0.5 mM concentration was added and then placed in a 37℃ shaking table for 3 h at 5000 ×g for 3 min.
4. Collect 50 mL bacteria into a 50 mL centrifuge tube. Discard the supernatant and freeze at -20℃.

Preparation of PDA (Potato Dextrose Agar) Medium

1. Clean the conical flask.
2. Weigh the PDA solid medium, according to the ratio of 200 mL: 9.2g, pour into the conical flask, add ultra-pure water, and seal with a sealing film.
3. Melt the solid in the microwave. Put it into the sterilizing pot, and set the sterilizing parameters: 115℃, 20 min.
4. The sterilized PDA medium is evenly poured into the petri dish.
5. Wait until solidified.
6. Sealing plate.

Expanding Cultivation of Rhizoctonia solani

1. Take out the refrigerated Rhizoctonia solani from the freezer and place it with the new petri dish on the sterilized clean bench.
2. Use a scoop to take a small piece of slime fungus medium from the Petri dish of Rhizoctonia solanum, place it into the new dish, and seal the edges of the two dishes with a sealing film.
3. Place the petri dish of Rhizoctonia solanum accepted into a constant temperature incubator at 28℃.

Crude Extraction of DsRNA by 75% Alcohol Precipitation

1. Take 50 mL of the induced bacterial solution, centrifuge at 4℃ 12000 ×g for 2 min, and discard the supernatant.
2. Add 2 mL 75% ethanol, blow and mix well, leave at room temperature for 5 min, centrifuge at 4℃ 8000 ×g, and then discard the supernatant.
3. The sediment was suspended with 500 µL of 150 mM NaCl solution and then left for 1 h at room temperature.
4. Centrifuge at 4℃ 12000Xg for 10 min, take 500 µL of supernatant for nucleic acid electrophoresis, and store at -80℃.

Crude Extraction of DsRNA by RNA-easy Method

1. Take 50mL bacterial solution, and centrifuge 5000 ×g for 3 min to collect bacterial precipitation.
2. Add 1 mL PBS suspension precipitation, transfer to the 1.5 mL enzyme-free tube, centrifuge 10000 ×g for 1 min centrifugation, and then discard supernatant for 1 min.
3. Add 500 µL of RNA-easy, shake, and leave at room temperature for 5 min, centrifuge 12000 ×g for 15 min.
4. Transfer the supernatant to a new 1.5 mL enzyme-free tube, add 500 µL of isopropyl alcohol, and leave at room temperature for 10 min. Centrifuge 12000 ×g for 10 min, discard the supernatant.
5. Centrifuge the 75% ethanol at 7500 ×g for 5 min, repeat washing the dsRNA.
6. Let the sediment stand at room temperature for 10 min and dry the retained ethanol.
7. Add 30 µL DEPC-treated water to dissolve the sediment, and store at -80℃.

DsRNA was Co-cultured with Rhizoctonia solani

1. Put 200 µL of sterilized ultra-pure water into the first plate of PDA culture medium.
2. Apply evenly with a spreader.
3. Prepare 200 µL of dsRNA solution with a concentration of 50 ng/µL into the second plate of the PDA culture medium.
4. Apply evenly with a spreader.
5. Dot the back of two plates of PDA culture medium to confirm the location of the bacteria.
6. Cut two same blocks of the Rhizoctonia solani.
7. Transfer two blocks into two plates of PDA culture medium.
8. Seal the medium with tape.
9. Weighing record.

DAB Staining of Tobacco

1. Dilute The extracted dsRNA to 50 ng/µl and spray on the tobacco leaves. Water was sprayed on the leaves of another tobacco plant as a control. Dry in the shade for 24 h;
2. Prepare 200 mL 10 mM Tris-HCl with pH 3.8.
3. Scrape half of the plate of Rhizoctonia acute with a spreader.
4. Mix in 1 mL DEPC-treated water.
5. Evenly apply 200 µl of Rhizoctonia solani solution on the left side of two pieces of tobacco.
6. Transfer bacteria to tobacco leaves and dry in the shade for 16 h.
7. 1.7 mg/mL DAB dyeing solution was prepared, 50 mL 10 mM Tris-HCl solution with pH 3.8, and put into a conical bottle; then 0.085 g DAB dyeing powder was dissolved in Tris-HCl. Wrapped the conical flask with tinfoil to avoid light. After dissolving, adjust to pH 5.7 with KOH.
8. Put the dyeing solution into a 50 mL measuring cylinder, take the whole tobacco leaves into the petri dish, and pour 25 mL of dyeing solution into the two plates to soak the leaves. Petri dishes are wrapped in tinfoil to avoid light.
9. Place on the horizontal shaker and shake for 23 h.
10. Pour away the dyeing solution, add 75% ethanol, heat in a water bath at 65℃ for 60 min, and replace the ethanol solution once at 30 min, until the chlorophyll is completely removed.
11. Drain the ethanol and wash it once with ultra-pure water, about 1 min.

Functional Verification of dsRNA Against Rhizoctonia solani in Maize

1. The extracted dsRNA was diluted to 50 ng/µl and sprayed on the maize leaves. Water was sprayed on the maize leaves as a control. Dry in the shade for 24 h.
2. Transfer the Rhizoctonia acuta into sterile water and apply it evenly on maize leaves. Observe the phenotype of maize leaves three days later.