Because the MP binds RNA in a sequence nonspecific manner [27],
it is probably designed to form a complex with RNA molecules in
its vicinity. Thus, while the MP binds to nearby viral RNA within
VRCs during infection, the MP may likely bind to the mRNA from
which it is translated under transient expression conditions. Upon
Agrobacterium tumefaciens-mediated transient expression, MP
reproduces its typical cellular accumulation and turnover pattern
seen during infection, thus forming mobile MP particles during
early transient expression stages. To determine if these particles
contain MP mRNA in addition to MP protein, we optimized the
MS2 RNA visualization system for MP mRNA detection in
N. benthamiana leaves. This allowed us to demonstrate that the
mobile MP particles indeed contain MP mRNA and that the
mRNA is also targeted together with MP to the PD [35].
Using the detection of MP mRNA as an example, we here
provide a detailed protocol for the MS2-based in vivo visualization
of mRNAs in plant cells. The protocol can be generally applied for
the study of mRNA localization and transport in plants. It combines the simple and reliable use of agroinfiltration for transient
expression in N. benthamiana leaves with the simplicity of Gateway
® cloning.
2 Materials
2.1 Cloning
of Plasmid Constructs
1. Standard thermal cycler (e.g., Biometra-Analytik Jena).
2. DNA polymerase for high-fidelity PCR amplification (e.g.,
Platinum™ Taq DNA Polymerase High Fidelity, Thermo
Fisher).
3. Agarose gel electrophoresis system.
4. Reagents for purification of DNA fragments from gels (e.g.,
PCR cleanup gel extraction kit, Macherey-Nagel).
5. PCR cloning system with plasmids and reagents (e.g., pGem
® -
T Easy, Promega).
6. E. coli cells for DNA cloning (e.g., DH5α), competent for
electro-transformation.
7. E. coli cells for the amplification of ccdB-expressing Gateway
®
plasmids (e.g., DB3.1), competent for electro-transformation.
8. Electroporator system (e.g., Gene Pulser Xcell, Bio-Rad) and
electroporation cuvettes.
9. LB liquid medium: 10 g/L Bacto-tryptone, 5 g/L yeast
extract, 85.6 mM NaCl, pH 7.0. Sterilize by autoclaving.
10. LB-agar plates: Add 15 g bacto-agar to 1.0 L of LB liquid
medium, sterilize by autoclaving, and pour plates while the
solution is still warm and not solidified.
108
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
it is probably designed to form a complex with RNA molecules in
its vicinity. Thus, while the MP binds to nearby viral RNA within
VRCs during infection, the MP may likely bind to the mRNA from
which it is translated under transient expression conditions. Upon
Agrobacterium tumefaciens-mediated transient expression, MP
reproduces its typical cellular accumulation and turnover pattern
seen during infection, thus forming mobile MP particles during
early transient expression stages. To determine if these particles
contain MP mRNA in addition to MP protein, we optimized the
MS2 RNA visualization system for MP mRNA detection in
N. benthamiana leaves. This allowed us to demonstrate that the
mobile MP particles indeed contain MP mRNA and that the
mRNA is also targeted together with MP to the PD [35].
Using the detection of MP mRNA as an example, we here
provide a detailed protocol for the MS2-based in vivo visualization
of mRNAs in plant cells. The protocol can be generally applied for
the study of mRNA localization and transport in plants. It combines the simple and reliable use of agroinfiltration for transient
expression in N. benthamiana leaves with the simplicity of Gateway
® cloning.
2 Materials
2.1 Cloning
of Plasmid Constructs
1. Standard thermal cycler (e.g., Biometra-Analytik Jena).
2. DNA polymerase for high-fidelity PCR amplification (e.g.,
Platinum™ Taq DNA Polymerase High Fidelity, Thermo
Fisher).
3. Agarose gel electrophoresis system.
4. Reagents for purification of DNA fragments from gels (e.g.,
PCR cleanup gel extraction kit, Macherey-Nagel).
5. PCR cloning system with plasmids and reagents (e.g., pGem
® -
T Easy, Promega).
6. E. coli cells for DNA cloning (e.g., DH5α), competent for
electro-transformation.
7. E. coli cells for the amplification of ccdB-expressing Gateway
®
plasmids (e.g., DB3.1), competent for electro-transformation.
8. Electroporator system (e.g., Gene Pulser Xcell, Bio-Rad) and
electroporation cuvettes.
9. LB liquid medium: 10 g/L Bacto-tryptone, 5 g/L yeast
extract, 85.6 mM NaCl, pH 7.0. Sterilize by autoclaving.
10. LB-agar plates: Add 15 g bacto-agar to 1.0 L of LB liquid
medium, sterilize by autoclaving, and pour plates while the
solution is still warm and not solidified.
108
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein