each clone in the infiltration mixture and the time after infiltration for observation must be empirically determined. Higher
concentration of the bacterial solutions used for agroinfiltration will lead to higher expression levels. For RNA labeling
experiments, high expression of NLS:MCP:GFP induces fluorescence background while low expression compromises the
sensitivity by which tagged RNA can be detected. For the
visualization of TMV MP mRNA we use bacterial solutions
with an OD 600 ¼ 0.5 for agrobacteria carrying pMDC32-RNA
or pMDC32-RNA-SL and an of OD 600 ¼ 0.1 for agrobacteria
carrying pB7-NLS-MCP:GFP.
6. The Agrobacterium-mediated transient expression level reaches
a maximum after 3–4 days following infiltration and then
decreases, which is, in part, due to posttranscriptional gene
silencing. A more sustained expression can be obtained by
co-expression of a viral silencing suppressor protein (e.g., P19
from tomato bushy stunt virus) [67]. In our experiments, we
rely on the co-expression of P19 to reach desirable levels of the
MSL-tagged clones [35]. Expression levels can also be
increased by inducing the A. tumefaciens vir genes. This can
be achieved by resuspending the bacteria in infiltration medium
(10 mM MES pH 5.6, 10 mM NaCl 2 , 150 μM acetosyringone)
followed by incubation for at least 2 h prior to infiltration.
7. In order to ensure a reliable MCP-mediated localization of the
mRNA under investigation, the background of the system
must be minimized. The MCP expression and localization
pattern must be evaluated in the presence of both MSL taggedor non-tagged mRNA.
8. The MP-containing mobile RNA granules shown in Fig. 1b
were detected as early as 30–36 h post-agroinfiltration. Imaging the motility of these dually labeled, mRNA-containing
granules required high-rate dual-color image acquisition over
extended time intervals. Initially, we achieved this by using a
standard epifluorescence microscope equipped with a dualview beam splitter (Optical Insights) for simultaneous imaging
of green and red fluorescence, and by image acquisition with a
high-speed charge-coupled device (CCD) camera (CoolSnap
HQ, Roper Scientific) [35]. Today, more modern dual-color
split-view systems are available that allow simultaneous
two-color imaging with two attached cameras (e.g., TuCam,
Oxford Instruments). The images shown in Fig. 1b were taken
with a confocal laser scanning microscope (Zeiss LSM780).
The frequency of image acquisition was increased by reducing
image size and resolution.
RNA Imaging in Plants Using MS2
117
concentration of the bacterial solutions used for agroinfiltration will lead to higher expression levels. For RNA labeling
experiments, high expression of NLS:MCP:GFP induces fluorescence background while low expression compromises the
sensitivity by which tagged RNA can be detected. For the
visualization of TMV MP mRNA we use bacterial solutions
with an OD 600 ¼ 0.5 for agrobacteria carrying pMDC32-RNA
or pMDC32-RNA-SL and an of OD 600 ¼ 0.1 for agrobacteria
carrying pB7-NLS-MCP:GFP.
6. The Agrobacterium-mediated transient expression level reaches
a maximum after 3–4 days following infiltration and then
decreases, which is, in part, due to posttranscriptional gene
silencing. A more sustained expression can be obtained by
co-expression of a viral silencing suppressor protein (e.g., P19
from tomato bushy stunt virus) [67]. In our experiments, we
rely on the co-expression of P19 to reach desirable levels of the
MSL-tagged clones [35]. Expression levels can also be
increased by inducing the A. tumefaciens vir genes. This can
be achieved by resuspending the bacteria in infiltration medium
(10 mM MES pH 5.6, 10 mM NaCl 2 , 150 μM acetosyringone)
followed by incubation for at least 2 h prior to infiltration.
7. In order to ensure a reliable MCP-mediated localization of the
mRNA under investigation, the background of the system
must be minimized. The MCP expression and localization
pattern must be evaluated in the presence of both MSL taggedor non-tagged mRNA.
8. The MP-containing mobile RNA granules shown in Fig. 1b
were detected as early as 30–36 h post-agroinfiltration. Imaging the motility of these dually labeled, mRNA-containing
granules required high-rate dual-color image acquisition over
extended time intervals. Initially, we achieved this by using a
standard epifluorescence microscope equipped with a dualview beam splitter (Optical Insights) for simultaneous imaging
of green and red fluorescence, and by image acquisition with a
high-speed charge-coupled device (CCD) camera (CoolSnap
HQ, Roper Scientific) [35]. Today, more modern dual-color
split-view systems are available that allow simultaneous
two-color imaging with two attached cameras (e.g., TuCam,
Oxford Instruments). The images shown in Fig. 1b were taken
with a confocal laser scanning microscope (Zeiss LSM780).
The frequency of image acquisition was increased by reducing
image size and resolution.
RNA Imaging in Plants Using MS2
117