3. Use cover slides with minimal thickness to obtain highresolution images.
4. Figure 2 shows the map of pBS-SL24 and pBS-SL48, which are
pBluescript-based plasmids that encode 8 and 16 copies of the
SL3 structure, and the enzymes that are used to excise the
DNA fragments encoding the SL repeats. Note that the fragments are oriented from KpnI to SacI (5
0 to 3
0 ) to fuse the SL
repeats to the RNA encoded by the target construct in correct
orientation. Although the number of SL3 structures used for
tagging the target mRNA may enhance fluorescent signals, a
higher SL3 copy number may also affect the stability and
transport of the SL-tagged mRNA.
5. Commercialized plasmid DNA extraction system (for extraction of plasmid DNA from bacteria) and gel extraction system
(for extraction of DNA fragments from agarose gels) are utilized for efficient cloning. Commercialized competent cells of
E. coli and A. tumefaciens strains are applied for
transformation.
6. Infiltrate the abaxial side of the leaves. Infiltration creates an
“infiltrated patch” on the leaves. Cells at the margin between
infiltrated and non-infiltrated tissues should be used for analysis
by confocal microscopy (Fig. 3b).
7. Watering should be avoided within 24 h after infiltration.
8. The removed leaf disks should be smaller than 5 Â 5 mm and
contain no large veins.
9. High expression of SL-tagged mobile mRNAs can interfere in
specific subcellular distribution of mobile mRNAs. The critical
mRNA abundance within a cell occurs within a short time after
agrobacteria infiltration. The cells at the boundary area display
lower expression of mobile mRNAs as compared to the cells in
the center of the infiltrated area. The boundary area represents
the margin between agroinfiltrated and non-infiltrated tissues,
which can be distinguished by observing GFP signals under
confocal microscopy. Cells located within the agroinfiltrated
patch exhibit extensive GFP fluorescence, whereas GFP fluorescence is not detected in cells outside the patch. The boundary area is the area where the two types of cells encounter. The
cells in boundary area usually exhibit moderate GFP fluorescence (bright nucleolus and faint fluorescence in the nucleoplasm). In our experience, selective PD targeting of mobile
mRNA is detected in the cells within or adjacent to the boundary area [28].
10. Considering the dynamic distribution of mobile mRNA,
images should be captured as fast as possible, by using a low
number of scans (e.g., 1) for each pixel line.
Plant mRNA Live-Cell Imaging Using MS2
153
4. Figure 2 shows the map of pBS-SL24 and pBS-SL48, which are
pBluescript-based plasmids that encode 8 and 16 copies of the
SL3 structure, and the enzymes that are used to excise the
DNA fragments encoding the SL repeats. Note that the fragments are oriented from KpnI to SacI (5
0 to 3
0 ) to fuse the SL
repeats to the RNA encoded by the target construct in correct
orientation. Although the number of SL3 structures used for
tagging the target mRNA may enhance fluorescent signals, a
higher SL3 copy number may also affect the stability and
transport of the SL-tagged mRNA.
5. Commercialized plasmid DNA extraction system (for extraction of plasmid DNA from bacteria) and gel extraction system
(for extraction of DNA fragments from agarose gels) are utilized for efficient cloning. Commercialized competent cells of
E. coli and A. tumefaciens strains are applied for
transformation.
6. Infiltrate the abaxial side of the leaves. Infiltration creates an
“infiltrated patch” on the leaves. Cells at the margin between
infiltrated and non-infiltrated tissues should be used for analysis
by confocal microscopy (Fig. 3b).
7. Watering should be avoided within 24 h after infiltration.
8. The removed leaf disks should be smaller than 5 Â 5 mm and
contain no large veins.
9. High expression of SL-tagged mobile mRNAs can interfere in
specific subcellular distribution of mobile mRNAs. The critical
mRNA abundance within a cell occurs within a short time after
agrobacteria infiltration. The cells at the boundary area display
lower expression of mobile mRNAs as compared to the cells in
the center of the infiltrated area. The boundary area represents
the margin between agroinfiltrated and non-infiltrated tissues,
which can be distinguished by observing GFP signals under
confocal microscopy. Cells located within the agroinfiltrated
patch exhibit extensive GFP fluorescence, whereas GFP fluorescence is not detected in cells outside the patch. The boundary area is the area where the two types of cells encounter. The
cells in boundary area usually exhibit moderate GFP fluorescence (bright nucleolus and faint fluorescence in the nucleoplasm). In our experience, selective PD targeting of mobile
mRNA is detected in the cells within or adjacent to the boundary area [28].
10. Considering the dynamic distribution of mobile mRNA,
images should be captured as fast as possible, by using a low
number of scans (e.g., 1) for each pixel line.
Plant mRNA Live-Cell Imaging Using MS2
153