4. Dilute the bacterial solutions for the agroinfiltration experiment. Create a bacterial “solution A” by combining pB7NLS-MCP:GFP-containing bacteria with pMDC32-RNASL-containing agrobacteria, and another bacterial “solution
B” by combining pB7-NLS-MCP:GFP-containing bacteria
with pMDC32-RNA-containing bacteria. Solution B is
required as control in the RNA visualization experiment. The
appropriate concentrations of the bacterial solutions to obtain a
good RNA imaging result must be empirically determined (see
Note 5).
5. To agroinfiltrate N. benthamiana plants with both solutions,
start by using a needle to puncture two holes into the youngest
fully expanded leaf at sites selected for agroinfiltration. Select
single sites on the left and on the right of the major vein.
Position a 1.0 mL or 2.5 mL syringe without needle and filled
with solution A onto the abaxial side of the punctured site on
the left and infiltrate the leaf blade with bacterial solution by
applying pressure onto the plunger. Upon infiltration with the
liquid, the infiltrated area acquires a darker color and thus can
be easily distinguished from the non-infiltrated, light-green
region. Infiltrate until the wetted, dark leaf area reaches the
major vein. Repeat the same procedure for infiltration of the
right side of the leaf blade with solution B.
6. Return the infiltrated plant(s) to their normal growing conditions in the greenhouse or growth chamber and incubate them
for the needed time until the gene constructs reach desired
expression levels suitable for microscopy (see Note 6).
3.3 Microscopy,
Sample Preparation,
and Conditions
for the Visualization
of MS2-Tagged RNA
1. Use a sharpened cork borer to excise samples from the leaf areas
infiltrated with solution A and solution B, respectively.
2. Place the leaf disks on microscopic slides and cover them with a
coverslip. The abaxial, infiltrated side of the disk should face the
coverslip. Fix the coverslip to the slide with tape. Fill the
airspace between coverslip and glass slide with sterile water.
3. To exchange the air present in intercellular spaces with water,
place the slides into a vacuum desiccator and evacuate the air
until the pressure reaches À0.8 to À1.0 bar. Subsequently,
slowly release the pressure to allow slow water entry. During
this process, the leaf disks should acquire a darker green color.
4. Analyze the leaf disk samples by microscopy. Start by using a
low-magnification objective to verify tissue integrity and to
determine if samples that were infiltrated with solution A and
solution B, respectively, express similar levels of NLS:MCP:
GFP. If the RNA under study encodes a fluorescently tagged
protein, the two samples expressing this protein from
MSL-tagged RNA and non-tagged RNA, respectively, should
show similar expression levels for this protein (see Note 7).
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Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
B” by combining pB7-NLS-MCP:GFP-containing bacteria
with pMDC32-RNA-containing bacteria. Solution B is
required as control in the RNA visualization experiment. The
appropriate concentrations of the bacterial solutions to obtain a
good RNA imaging result must be empirically determined (see
Note 5).
5. To agroinfiltrate N. benthamiana plants with both solutions,
start by using a needle to puncture two holes into the youngest
fully expanded leaf at sites selected for agroinfiltration. Select
single sites on the left and on the right of the major vein.
Position a 1.0 mL or 2.5 mL syringe without needle and filled
with solution A onto the abaxial side of the punctured site on
the left and infiltrate the leaf blade with bacterial solution by
applying pressure onto the plunger. Upon infiltration with the
liquid, the infiltrated area acquires a darker color and thus can
be easily distinguished from the non-infiltrated, light-green
region. Infiltrate until the wetted, dark leaf area reaches the
major vein. Repeat the same procedure for infiltration of the
right side of the leaf blade with solution B.
6. Return the infiltrated plant(s) to their normal growing conditions in the greenhouse or growth chamber and incubate them
for the needed time until the gene constructs reach desired
expression levels suitable for microscopy (see Note 6).
3.3 Microscopy,
Sample Preparation,
and Conditions
for the Visualization
of MS2-Tagged RNA
1. Use a sharpened cork borer to excise samples from the leaf areas
infiltrated with solution A and solution B, respectively.
2. Place the leaf disks on microscopic slides and cover them with a
coverslip. The abaxial, infiltrated side of the disk should face the
coverslip. Fix the coverslip to the slide with tape. Fill the
airspace between coverslip and glass slide with sterile water.
3. To exchange the air present in intercellular spaces with water,
place the slides into a vacuum desiccator and evacuate the air
until the pressure reaches À0.8 to À1.0 bar. Subsequently,
slowly release the pressure to allow slow water entry. During
this process, the leaf disks should acquire a darker green color.
4. Analyze the leaf disk samples by microscopy. Start by using a
low-magnification objective to verify tissue integrity and to
determine if samples that were infiltrated with solution A and
solution B, respectively, express similar levels of NLS:MCP:
GFP. If the RNA under study encodes a fluorescently tagged
protein, the two samples expressing this protein from
MSL-tagged RNA and non-tagged RNA, respectively, should
show similar expression levels for this protein (see Note 7).
114
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein