2. A. tumefaciens carrying a plasmid driving abundant and ubiquitous expression of a protein of interest, genetically fused to a
red fluorescent protein (e.g., tagRFP or mCherry).
3. Inoculum of the virus of interest, here in the form of
A. tumefaciens carrying a virus-encoding plasmid for agroinfection.
4. 50 mL Falcon tubes.
5. Centrifuge able to spin 50 mL Falcon tubes at 3000 Â g.
6. 200 mM Acetosyringone.
7. MMA buffer: 10 mM MES pH 5.6, 10 mM MgCl 2 , 200 μM
acetosyringone.
8. Spectrophotometer to measure optical density at 600 nm
(OD 600 ).
9. Syringes without needle.
10. Microscopy glass slides.
11. Coverslips.
12. Vacuum pump.
13. Confocal laser fluorescence microscope.
3 Methods
3.1 dsRNA
Pull-Down
All steps should be performed at 4
C or on ice, and as rapidly as
possible while pipetting and handling gently.
1. Harvest 0.5 g or more of systemically infected leaves from
GFP- and B2:GFP-expressing plants and place them into collection tubes in liquid nitrogen. Break the leaves into a coarse
powder and store at À80
C.
2. Place 0.1 g of tissue powder into a mortar pre-chilled with
liquid nitrogen (keep an aliquot of tissue powder aside for use
as total tissue sample during later analysis; see Subheading 3.2)
(see Note 1). Grind the tissue to a fine powder with a pestle,
add 0.5 mL cold lysis buffer, homogenize with pestle, and add
again 0.5 mL cold lysis buffer. Transfer the homogenate to a
1.5 mL Eppendorf and incubate for 10 min on a rotating wheel
at 4
C.
3. Centrifuge at 4
C for 5–10 min at 12,000 Â g and transfer the
supernatant to a new tube (see Note 2). Transfer 60 μL of the
solution into fresh tube and set aside on ice. This aliquot will be
used as “input” in the later analysis of the pull-down
experiment.
4. Add anti-GFP magnetic beads to the main solution. The quantity of beads depends on the product used and on
Pull-Down of Viral Double-Stranded RNA
313
red fluorescent protein (e.g., tagRFP or mCherry).
3. Inoculum of the virus of interest, here in the form of
A. tumefaciens carrying a virus-encoding plasmid for agroinfection.
4. 50 mL Falcon tubes.
5. Centrifuge able to spin 50 mL Falcon tubes at 3000 Â g.
6. 200 mM Acetosyringone.
7. MMA buffer: 10 mM MES pH 5.6, 10 mM MgCl 2 , 200 μM
acetosyringone.
8. Spectrophotometer to measure optical density at 600 nm
(OD 600 ).
9. Syringes without needle.
10. Microscopy glass slides.
11. Coverslips.
12. Vacuum pump.
13. Confocal laser fluorescence microscope.
3 Methods
3.1 dsRNA
Pull-Down
All steps should be performed at 4
C or on ice, and as rapidly as
possible while pipetting and handling gently.
1. Harvest 0.5 g or more of systemically infected leaves from
GFP- and B2:GFP-expressing plants and place them into collection tubes in liquid nitrogen. Break the leaves into a coarse
powder and store at À80
C.
2. Place 0.1 g of tissue powder into a mortar pre-chilled with
liquid nitrogen (keep an aliquot of tissue powder aside for use
as total tissue sample during later analysis; see Subheading 3.2)
(see Note 1). Grind the tissue to a fine powder with a pestle,
add 0.5 mL cold lysis buffer, homogenize with pestle, and add
again 0.5 mL cold lysis buffer. Transfer the homogenate to a
1.5 mL Eppendorf and incubate for 10 min on a rotating wheel
at 4
C.
3. Centrifuge at 4
C for 5–10 min at 12,000 Â g and transfer the
supernatant to a new tube (see Note 2). Transfer 60 μL of the
solution into fresh tube and set aside on ice. This aliquot will be
used as “input” in the later analysis of the pull-down
experiment.
4. Add anti-GFP magnetic beads to the main solution. The quantity of beads depends on the product used and on
Pull-Down of Viral Double-Stranded RNA
313