5. Use a high-magnification objective to investigate the leaf disks
that were infiltrated with solution A to visualize the cytoplasmic
localization of the MSL-tagged RNA labeled by GFP fluorescence emitted from NLS:MCP:GFP bound to it. Specific cytoplasmic fluorescent signals that are due to specific binding of
NLS:MCP:GFP to the MSL repeats in MSL-tagged RNA
should be absent in the control samples that were infiltrated
with solution B and in which the RNA lacks the MSL sequences
required for NLS:MCP:GFP binding. Acquire images from
cells expressing both samples and time-lapse videos at high
sampling rates to capture the movements of detected RNA
granules. Signal detection is facilitated in the abaxial epidermal
pavement cells in which most of the volume is occupied by the
vacuole, which compresses the cytoplasm toward the plasma
membrane and cell wall.
6. Use ImageJ or similar software for image acquisition and analysis (e.g., colocalization, particle tracking, velocity measurement). Observations should finish within 30 min after leaf
disks were excised. After this time, new samples need to be
prepared.
7. Images of RNA granules formed by the mRNA of the TMV
MP are seen in Fig. 1. In views focusing on the lateral cell walls
of epidermal cells, the cytoplasm is condensed to a thin line
along the wall, and MP:RFP fluorescence is detected at PD
(Fig. 1a). Views focusing on the cortical cytoplasm just beneath
the upper epidermal cell wall allow the observation of thin
layers of cytoplasm, where the ER-actin network and the cortical MT array share the same focal plane below the plasma
membrane. Here, RNA granules are detected and their lateral
movement can be visualized (Fig. 1b) (see Note 8).
4 Notes
1. pSL-MS2 plasmids containing different numbers of MSL copies as well as plasmids expressing different MCP variants are
available from the plasmid repository Addgene (https://www.
addgene.org/Robert_Singer/).
2. pMDC32 is available from TAIR (https://www.arabidopsis.
org/servlets/TairObject?type¼vector&id¼501100106).
3. Gateway
® cloning is a versatile technology that allows the fast
and precise exchange of DNA material, based on recombination. An entry clone contains your DNA of interest flanked by
attL sequences, which allow recombination in a LR™ reaction
with the attR site of the destination vector, to obtain the
desired final expression plasmid.
RNA Imaging in Plants Using MS2
115
that were infiltrated with solution A to visualize the cytoplasmic
localization of the MSL-tagged RNA labeled by GFP fluorescence emitted from NLS:MCP:GFP bound to it. Specific cytoplasmic fluorescent signals that are due to specific binding of
NLS:MCP:GFP to the MSL repeats in MSL-tagged RNA
should be absent in the control samples that were infiltrated
with solution B and in which the RNA lacks the MSL sequences
required for NLS:MCP:GFP binding. Acquire images from
cells expressing both samples and time-lapse videos at high
sampling rates to capture the movements of detected RNA
granules. Signal detection is facilitated in the abaxial epidermal
pavement cells in which most of the volume is occupied by the
vacuole, which compresses the cytoplasm toward the plasma
membrane and cell wall.
6. Use ImageJ or similar software for image acquisition and analysis (e.g., colocalization, particle tracking, velocity measurement). Observations should finish within 30 min after leaf
disks were excised. After this time, new samples need to be
prepared.
7. Images of RNA granules formed by the mRNA of the TMV
MP are seen in Fig. 1. In views focusing on the lateral cell walls
of epidermal cells, the cytoplasm is condensed to a thin line
along the wall, and MP:RFP fluorescence is detected at PD
(Fig. 1a). Views focusing on the cortical cytoplasm just beneath
the upper epidermal cell wall allow the observation of thin
layers of cytoplasm, where the ER-actin network and the cortical MT array share the same focal plane below the plasma
membrane. Here, RNA granules are detected and their lateral
movement can be visualized (Fig. 1b) (see Note 8).
4 Notes
1. pSL-MS2 plasmids containing different numbers of MSL copies as well as plasmids expressing different MCP variants are
available from the plasmid repository Addgene (https://www.
addgene.org/Robert_Singer/).
2. pMDC32 is available from TAIR (https://www.arabidopsis.
org/servlets/TairObject?type¼vector&id¼501100106).
3. Gateway
® cloning is a versatile technology that allows the fast
and precise exchange of DNA material, based on recombination. An entry clone contains your DNA of interest flanked by
attL sequences, which allow recombination in a LR™ reaction
with the attR site of the destination vector, to obtain the
desired final expression plasmid.
RNA Imaging in Plants Using MS2
115