6. Use the LR™ clonase mix (follow the manufacturer’s instructions) to perform separate LR™ reactions between the entry
vector now containing the DNA of your RNA of interest and
both (1) pMDC32 and (2) pMDC32-GW-12xMSL (see Subheading 3.1.1, step 10) to obtain the expression vectors
pMDC32-RNA and pMDC32-RNA-12xMSL, respectively
(follow the manufacturer’s instructions). Transform E. coli
cells and select them on LB agar plates with antibiotic selection
(50 μg/mL kanamycin).
7. Start 4–5 liquid cultures from isolated bacterial colonies and
grow them overnight under antibiotic selection (50 μg/mL
kanamycin) at 37
C with agitation (180–200 rpm).
8. Isolate the plasmids using a plasmid purification kit and verify
proper vector assembly by DNA sequencing (for our destination vectors pMDC32-MP:mRFP and pMDC32-MP:mRFP12xMSL we used internal fragment primers MP-GV1rev and
mRFPfw).
3.2 AgrobacteriumMediated Transient
Expression
of MSL-Tagged RNA
and Fluorescent MCP
in N. benthamiana
Leaves
The Gateway
® system destination vectors used in this protocol are
binary vectors suitable for transformation of Agrobacteria and
Agrobacterium-mediated transformation of plants. Infiltration of
plant leaf tissues with diluted agrobacteria containing the destination expression vector of interest is a flexible and reliable method
for the expression of foreign proteins in N. benthamiana leaves. By
mixing two Agrobacterium cultures harboring two different expression vectors, two proteins of interest can be simultaneously
expressed within the cells of the infiltrated leaf area [66]. Here,
we apply this co-transfection method for expression of the
MSL-tagged RNA together with fluorescent protein-tagged MCP
in the same cells.
1. Transform
separate
aliquots
of
electrocompetent
A. tumefaciens GV3101 cells with plasmids pMDC32-RNA,
pMDC32-RNA-12xMSL, and pB7-NLS-MCP:GFP [35],
respectively, by electroporation and grow colonies for 48 h at
28
C on LB agar plates containing rifampicin and gentamicin
(both at 50 μg/mL) and either (1) kanamycin (50 μg/mL) for
pMDC32 vectors or (2) spectinomycin (50 μg/mL) for
pB7-NLS-MCP:GFP.
2. Use individual colonies to start liquid cultures in 50 mL falcon
tubes containing 5.0 mL of LB medium and under antibiotic
selection as used for the plates in the previous step. Incubate
the cultures overnight at 28
C with agitation (180–200 rpm).
3. Harvest the bacteria by centrifugation at 5000 Â g for 5 min
and resuspend the bacterial pellets in sterile distilled water.
Determine the optical density of the bacterial solution at
600 nm.
RNA Imaging in Plants Using MS2
113
vector now containing the DNA of your RNA of interest and
both (1) pMDC32 and (2) pMDC32-GW-12xMSL (see Subheading 3.1.1, step 10) to obtain the expression vectors
pMDC32-RNA and pMDC32-RNA-12xMSL, respectively
(follow the manufacturer’s instructions). Transform E. coli
cells and select them on LB agar plates with antibiotic selection
(50 μg/mL kanamycin).
7. Start 4–5 liquid cultures from isolated bacterial colonies and
grow them overnight under antibiotic selection (50 μg/mL
kanamycin) at 37
C with agitation (180–200 rpm).
8. Isolate the plasmids using a plasmid purification kit and verify
proper vector assembly by DNA sequencing (for our destination vectors pMDC32-MP:mRFP and pMDC32-MP:mRFP12xMSL we used internal fragment primers MP-GV1rev and
mRFPfw).
3.2 AgrobacteriumMediated Transient
Expression
of MSL-Tagged RNA
and Fluorescent MCP
in N. benthamiana
Leaves
The Gateway
® system destination vectors used in this protocol are
binary vectors suitable for transformation of Agrobacteria and
Agrobacterium-mediated transformation of plants. Infiltration of
plant leaf tissues with diluted agrobacteria containing the destination expression vector of interest is a flexible and reliable method
for the expression of foreign proteins in N. benthamiana leaves. By
mixing two Agrobacterium cultures harboring two different expression vectors, two proteins of interest can be simultaneously
expressed within the cells of the infiltrated leaf area [66]. Here,
we apply this co-transfection method for expression of the
MSL-tagged RNA together with fluorescent protein-tagged MCP
in the same cells.
1. Transform
separate
aliquots
of
electrocompetent
A. tumefaciens GV3101 cells with plasmids pMDC32-RNA,
pMDC32-RNA-12xMSL, and pB7-NLS-MCP:GFP [35],
respectively, by electroporation and grow colonies for 48 h at
28
C on LB agar plates containing rifampicin and gentamicin
(both at 50 μg/mL) and either (1) kanamycin (50 μg/mL) for
pMDC32 vectors or (2) spectinomycin (50 μg/mL) for
pB7-NLS-MCP:GFP.
2. Use individual colonies to start liquid cultures in 50 mL falcon
tubes containing 5.0 mL of LB medium and under antibiotic
selection as used for the plates in the previous step. Incubate
the cultures overnight at 28
C with agitation (180–200 rpm).
3. Harvest the bacteria by centrifugation at 5000 Â g for 5 min
and resuspend the bacterial pellets in sterile distilled water.
Determine the optical density of the bacterial solution at
600 nm.
RNA Imaging in Plants Using MS2
113