1. Use single colonies of transformed A. tumefaciens bacteria
containing the abovementioned plasmids as well as bacteria
containing pCAMBIA1390-35S-MS2 FD -GFP to separately
inoculate 2 mL LB medium in test tubes. Incubate the bacterial
cultures at 28
C on a shaker set to 220 rpm for 24 h.
2. Transfer 200 μL of each overnight culture into a 125 mL glass
flask containing 10 mL inoculation medium. Incubate the
flasks at 28
C on a shaker set to 220 rpm overnight.
3. Transfer the medium with bacteria from each flask to a 50 mL
Falcon tube. Collect bacteria by centrifugation for 5 min at
9000 Â g in the cold (4
C). Resuspend the bacterial pellets in
3 mL infiltration medium and adjust the concentration of
bacteria to OD 600 ¼ 1 with infiltration medium.
4. Incubate the bacteria in the infiltration solution for 1 h at room
temperature.
5. Set up the following mixtures by mixing equal volumes (1 mL
for each solution) of the bacteria-containing infiltration solutions in a 15 mL Falcon tube:
(a) pCAMBIA1390-35S-MS2 FD -GFP
with
infiltration
medium.
(b) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-FT SL24 .
(c) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-FT.
(d) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-RFP SL24 .
(e) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-RFP.
6. Use a 5 mL syringe without needle to infiltrate the abaxial side
of Nicotiana benthamiana leaves (see Note 6) (Fig. 3a). Use
4-week-old N. benthamiana plants and infiltrate the fifth to
seventh leaves (counted from the bottom of the plants). Incubate the infiltrated plants in a growth chamber set to 28
C and
a 14-h/10-h light/dark cycle for 24–48 h before analysis by
confocal microscopy (see Note 7).
3.3 Imaging
1. Use a 10 cm ophthalmic scissor to remove a leaf disk (see
Note 8) from the rim of the agroinfiltrated patch (Fig. 3b).
2. Place the leaf disk on a microscope glass slide with the adaxial
side facing the glass. Use a plastic dropper to apply an appropriate amount of sterilized-distilled water onto the abaxial side
of leaf disk. Cover the leaf disk with a cover slide. Gently press
the cover slide and remove excess water with Kimwipes.
Plant mRNA Live-Cell Imaging Using MS2
151
containing the abovementioned plasmids as well as bacteria
containing pCAMBIA1390-35S-MS2 FD -GFP to separately
inoculate 2 mL LB medium in test tubes. Incubate the bacterial
cultures at 28
C on a shaker set to 220 rpm for 24 h.
2. Transfer 200 μL of each overnight culture into a 125 mL glass
flask containing 10 mL inoculation medium. Incubate the
flasks at 28
C on a shaker set to 220 rpm overnight.
3. Transfer the medium with bacteria from each flask to a 50 mL
Falcon tube. Collect bacteria by centrifugation for 5 min at
9000 Â g in the cold (4
C). Resuspend the bacterial pellets in
3 mL infiltration medium and adjust the concentration of
bacteria to OD 600 ¼ 1 with infiltration medium.
4. Incubate the bacteria in the infiltration solution for 1 h at room
temperature.
5. Set up the following mixtures by mixing equal volumes (1 mL
for each solution) of the bacteria-containing infiltration solutions in a 15 mL Falcon tube:
(a) pCAMBIA1390-35S-MS2 FD -GFP
with
infiltration
medium.
(b) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-FT SL24 .
(c) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-FT.
(d) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-RFP SL24 .
(e) pCAMBIA1390-35S-MS2 FD -GFP with pCAMBIA139035S-RFP.
6. Use a 5 mL syringe without needle to infiltrate the abaxial side
of Nicotiana benthamiana leaves (see Note 6) (Fig. 3a). Use
4-week-old N. benthamiana plants and infiltrate the fifth to
seventh leaves (counted from the bottom of the plants). Incubate the infiltrated plants in a growth chamber set to 28
C and
a 14-h/10-h light/dark cycle for 24–48 h before analysis by
confocal microscopy (see Note 7).
3.3 Imaging
1. Use a 10 cm ophthalmic scissor to remove a leaf disk (see
Note 8) from the rim of the agroinfiltrated patch (Fig. 3b).
2. Place the leaf disk on a microscope glass slide with the adaxial
side facing the glass. Use a plastic dropper to apply an appropriate amount of sterilized-distilled water onto the abaxial side
of leaf disk. Cover the leaf disk with a cover slide. Gently press
the cover slide and remove excess water with Kimwipes.
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151