Experiment Summary

When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double- stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purified dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.

Materials and Reagents

A. Plasmid DNAs for Production of Recombinant Proteins

1. pGST-DRB4*-CaL

B. Preparation of Recombinant Proteins

1. Escherichia coli Rosetta competent cells.
2. LB medium.
3. 100 mg/ml carbenicillin dissolved in water.
4. 0.1 M isopropyl β- D-1-thiogalactopyranoside (IPTG) dissolved in water and filter-sterilized. Store at -25 °C.
5. P buffer: 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA. Store at 4 °C.
6. 10 mg/ml RNase A.
7. Glutathione Sepharose 4B. Store at 4 °C.
8. Glutathione Sepharose resin equilibrated with P buffer.
9. Elution buffer: 50 mM reduced glutathione, 50 mM Tris-HCl (pH 8.0). Prepare immediately before use or store at -25 °C in aliquots for single experiments.
10. SDS-PAGE.
11. Amicon Ultra-15 centrifugal filter units.
12. Dialysis tubing.
13. 0.1 M sodium phosphate buffer (pH 7.0).

C. In Vitro Transcription for Preparation of Artificial dsRNA

1. Template DNA
2. A kit for gel purification of PCR-amplified DNA fragments.
3. CUGA 7in vitro transcription kit
4. Phenol-chloroform-isoamyl alcohol (25:24:1).
5. Ethanol.
6. 3 M sodium acetate (pH 5.2).
7. TURBO DNase I, RNase-free.
8. RNase-free TE buffer.

D. Total RNA Extraction

1. D solution.
2. Acid phenol.
3. 3 M sodium acetate (pH 5.2).
4. Chloroform.
5. 2-propanol.
6. RNase-free TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA.
7. A tissue disruptor such as a Micro Smash system.
8. Screw-cap tubes for tissue disruption.
9. Stainless steel beads: 2 beads/tube, 5 mm diameter.

E. dsRNA Isolation

1. dRBB (dsRNA binding buffer).
2. dRBB containing 0.1 % Tween 20. Store at room temperature.
3. Glutathione Sepharose 4B suspended in dRBB containing 0.1 % BSA. Store at 4 °C.
4. TE buffer containing 0.5 % SDS, 5 mM EDTA, and loading dye. Store at room temperature.
5. Ethachinmate.

Procedure

A. Expression of GST-DRB4* Protein in E. coli

1. Transform E. coli Rosetta with pGST-DRB4*-CaL. Inoculate 2 ml LB liquid medium containing 100 mg/l carbenicillin with a single colony of the transformant and culture overnight at 37 °C with vigorous shaking. Dilute the overnight culture in 200 ml of fresh LB liquid medium containing 100 mg/l carbenicillin. Culture at 30 °C with vigorous shaking until the optical density reaches about 0.5.
2. Add IPTG to a final concentration of 1 mM to induce GSTDRB4* protein expression and culture for an additional 1.5 h at 30 °C with vigorous shaking.

B. Purification of GST-DRB4*

1. Collect bacterial cells by centrifugation for 5 min at 5,000 × g at 4 °C and suspend in 10 ml (1/20 volume of the culture) of P buffer containing 100 mg/l RNase A to remove RNA.
2. Sonicate the cells to complete cell lysis and centrifuge for 30 min at 20,000 × g at 4 °C to remove cell debris.
3. Transform E. coli Rosetta with pGST-DRB4*-CaL. Inoculate 2 ml LB liquid medium containing 100 mg/l carbenicillin with a single colony of the transformant and culture overnight at 37 °C with vigorous shaking. Dilute the overnight culture in 200 ml of fresh LB liquid medium containing 100 mg/l carbenicillin. Culture at 30 °C with vigorous shaking until the optical density reaches about 0.5.
4. Transfer the resin to an appropriate column and wash the resin with P buffer. Check the protein in the flowthrough by measuring the absorbance at 280 nm (A 280) or staining with Coomassie Brilliant Blue solution. Continue washing until no protein is detectable in the flowthrough.
5. Elute GST-DRB4* protein with 5 ml elution buffer and collect each 0.5 ml fraction.
6. Analyze the fractions by SDS-PAGE and collect fractions that contain GST-DRB4*.
7. Check the A₂₈₀ and roughly estimate the protein yield. Concentration of proteins is calculated as A₂₈₀ = 1.0 being equivalent to 1 mg/ml.
8. Concentrate the collected solutions to more than 2 mg/ml using Amicon Ultra-15 centrifugal fi lter units.
9. Dialyze against 0.1 M phosphate buffer, pH 7.0, for at least 2 days with three buffer changes.
10. Check the A₂₈₀ and A₂₆₀. The A₂₈₀/ A₂₆₀ ratio should be greater than 1.5. A protein concentration greater than 1 mg/ml is recommended.
11. Store the proteins at -25 °C in aliquots.

C. Preparation of Artificial dsRNA for Control Experiment

Artificial dsRNA for control experiments is prepared by an in vitro transcription of mixed PCR-amplified DNA fragments containing a single T7 promoter at the end PCR products should be gel-purified using a kit.

1. Set up in vitro transcription reaction as follows:
   4 μl 5× transcription buffer
   2 μl 0.1 M DTT
   1.5 μl each 100 mM CTP, UTP, GTP, and ATP
   1 μl CUGA 7 enzyme solution
   0.05-0.25 pmol of templates obtained as PCR products (sense: antisense = 1:1)
   RNase-free water up to 20 μl.
2. Incubate the reaction mixture at 37 °C for 2 h.
3. Denature RNA in the transcription reaction mixture at 75 °C for 15 min. Gradually cool the mixture to 20 °C at a rate of −0.5 °C/min to allow dsRNA formation.
4. Add DNase I and incubate at 37 °C for 30 min to remove template DNA.
5. Purify dsRNA by phenol-chloroform extraction and ethanol precipitation. Dissolve dsRNA in RNase-free TE buffer and store at -80 °C.

D. Total RNA Extraction from Virus-Infected Plants

1. Place the plant tissue sample (100 mg) into a 2-ml screw-cap microtube together with two stainless steel beads, tightly seal the cap, and place the tube in liquid nitrogen. Grind the sample to fi ne powder using a cell disrupter.
2. Add 500 μl D solution and mix thoroughly by vigorous shaking.
3. Add 500 μl acid phenol and 50 μl 3 M sodium acetate (pH 5.2), and centrifuge at 15,000 × g for 10 min at 4 °C.
4. Collect supernatant in a new tube, add 0.1 ml chloroform, mix by vigorous shaking, and incubate for 10 min on ice.
5. Centrifuge at 15,000 × g for 5 min and collect the upper aqueous phase in a new tube.
6. Add an equal volume of chloroform and mix by vigorous shaking.
7. Centrifuge at 15,000 × g for 5 min and collect the upper aqueous phase in a new tube.
8. Precipitate RNA by adding an equal volume of 2-propanol, shaking vigorously and centrifuging at 15,000 × g for 10 min at 4 °C. Wash RNA pellet with 70 % ethanol, air-dry, and dissolve in TE buffer. The RNA preparation can be used for dsRNA isolation or stored at -80 °C.

E. dsRNA Isolation

1. Add total RNA from the plants, or the artificial dsRNA for a control experiment, (up to one-tenth the volume of dRBB) to 0.5-1 ml dRBB in a 1.5 ml tube and mix thoroughly.
2. Add 10-20 μg GST-DRB4* to samples (up to 1/50 volume). Incubate at room temperature for 10 min.
3. Add 60 μl of a 35 % slurry of Glutathione Sepharose resin suspended in dRBB containing 0.1 % BSA (added to suppress nonspecific binding of RNA to the resin). Rotate the tube for 30 min to 1 h at 4 °C.
4. Pellet the resin by centrifugation at 2,000 × g for 5 s, remove supernatant, and resuspend the resin in 1 ml dRBB containing 0.1 % Tween 20; repeat this washing step three times.
5. Pellet the resin by centrifugation at 2,000 × g for 5 s, remove supernatant, and resuspend the resin in 1 ml dRBB without 0.1 % Tween 20; repeat this washing step twice. After the second wash with dRBB alone, briefly centrifuge the resin and remove as much of the supernatant as possible using a micropipette.
6. For gel electrophoresis analysis alone, add 20 μl TE buffer containing 0.5 % SDS, 5 mM EDTA, and loading dye. Incubate for 5 min, pellet the resin by centrifugation and load the supernatant in the agarose gel for electrophoresis.
7. For the analysis of dsRNA by PCR and/or sequencing, add 100 μl TE buffer containing 0.5 % SDS, 5 mM EDTA and incubate for 5 min at room temperature.
8. Centrifuge at 2,000 × g for 10 s and collect supernatant in a new tube.
9. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), mix vigorously and centrifuge at 15,000 × g for 2 min.
10. Collect supernatant in a new tube, add an equal volume of chloroform-isoamyl alcohol (24:1), mix vigorously and centrifuge at 15,000 × g for 2 min.
11. Collect supernatant in a new tube and add 1 μl ethachinmate, 1/10 volume 3 M sodium acetate (pH 5.2), and 2–2.5 volumes ethanol.
12. Centrifuge at 15,000 × g for 15 min. Wash the pellet with 70 % ethanol and air-dry for 10 min. Add appropriate buffer for the downstream experiment.