15. Verify the presence, number, and orientation of 2xcsy stemloop tags by DNA sequencing with primer PVX5539F (see
Note 8).
3.3 Inoculation of N.
benthamiana with
Tagged PVX by
Microprojectile
Bombardment
1. Thoroughly vortex the ethanol-suspended gold particles until
the gold is evenly distributed throughout the solution, visible
by the dark color.
2. Immediately pipette 7 μL of the gold suspension into a clean
1.5 mL tube.
3. Add 3 μL of pTRA.PVX.2xcsy.mCherry-2A-CP plasmid DNA
and vortex immediately and thoroughly.
4. Add 12 μL of ethanol and vortex immediately and thoroughly.
5. Set the nitrogen gas pressure on the gene gun to 20–25 psi at
the regulator valve, and the intensity dial of the trigger to 20.
6. Hold the tube with the DNA-gold mixture by the side with the
lid open and vortex. While vortexing, pipet 5 μL DNA-gold
mixture and dispense them onto the grid of a Swinnex 13 mm
syringe filter holder. Screw the top onto the filter holder, and
then screw the filter holder into the nozzle of the gene gun.
7. Place a N. benthamiana leaf below the gene gun nozzle so that
the lower opening of the cartridge sits 2–3 cm above the leaf.
Gently turn the leaf to make the lower surface face upward.
8. Bombard the lower surface twice, moving the leaf a few centimeters in between.
9. For inoculating additional leaves, repeat with a fresh 5 μL
aliquot of DNA-gold mixture for each leaf.
3.4 Agro-Infiltration
of N. benthamiana
with Csy4∗
Expression Plasmids
1. On the same day as the PVX inoculation (Subheading 3.3),
inoculate 5 mL LB liquid medium containing 100 μg/mL
spectinomycin and 50 μg/mL rifampicin with A. tumefaciens
AGL1 transformed with either pGWB405.Csy4∗-GFP or
pGWB405.Csy4∗-NLS-GFP from a glycerol stock (from Subheading 3.1, step 24) (see Note 9), and incubate at 28
C with
shaking for 2–3 days.
2. Also inoculate 5 mL LB liquid medium containing 50 μg/mL
kanamycin and 50 μg/mL rifampicin with A. tumefaciens
AGL1 transformed with pDGB3alpha2_35S:P19:Tnos
(GB1203) from a glycerol stock, and incubate as above.
3. Centrifuge the liquid cultures at 3000 Â g for 15 min to pellet
the cells.
4. Decant the liquid medium and resuspend each cell pellet in
2 mL of infiltration buffer.
RNA Imaging with Csy4
169
Note 8).
3.3 Inoculation of N.
benthamiana with
Tagged PVX by
Microprojectile
Bombardment
1. Thoroughly vortex the ethanol-suspended gold particles until
the gold is evenly distributed throughout the solution, visible
by the dark color.
2. Immediately pipette 7 μL of the gold suspension into a clean
1.5 mL tube.
3. Add 3 μL of pTRA.PVX.2xcsy.mCherry-2A-CP plasmid DNA
and vortex immediately and thoroughly.
4. Add 12 μL of ethanol and vortex immediately and thoroughly.
5. Set the nitrogen gas pressure on the gene gun to 20–25 psi at
the regulator valve, and the intensity dial of the trigger to 20.
6. Hold the tube with the DNA-gold mixture by the side with the
lid open and vortex. While vortexing, pipet 5 μL DNA-gold
mixture and dispense them onto the grid of a Swinnex 13 mm
syringe filter holder. Screw the top onto the filter holder, and
then screw the filter holder into the nozzle of the gene gun.
7. Place a N. benthamiana leaf below the gene gun nozzle so that
the lower opening of the cartridge sits 2–3 cm above the leaf.
Gently turn the leaf to make the lower surface face upward.
8. Bombard the lower surface twice, moving the leaf a few centimeters in between.
9. For inoculating additional leaves, repeat with a fresh 5 μL
aliquot of DNA-gold mixture for each leaf.
3.4 Agro-Infiltration
of N. benthamiana
with Csy4∗
Expression Plasmids
1. On the same day as the PVX inoculation (Subheading 3.3),
inoculate 5 mL LB liquid medium containing 100 μg/mL
spectinomycin and 50 μg/mL rifampicin with A. tumefaciens
AGL1 transformed with either pGWB405.Csy4∗-GFP or
pGWB405.Csy4∗-NLS-GFP from a glycerol stock (from Subheading 3.1, step 24) (see Note 9), and incubate at 28
C with
shaking for 2–3 days.
2. Also inoculate 5 mL LB liquid medium containing 50 μg/mL
kanamycin and 50 μg/mL rifampicin with A. tumefaciens
AGL1 transformed with pDGB3alpha2_35S:P19:Tnos
(GB1203) from a glycerol stock, and incubate as above.
3. Centrifuge the liquid cultures at 3000 Â g for 15 min to pellet
the cells.
4. Decant the liquid medium and resuspend each cell pellet in
2 mL of infiltration buffer.
RNA Imaging with Csy4
169