3.3.2 Generation
of Binary 3
0 UTR or ORF
Sensors
1. Set up LR recombination reactions from the 3
0 UTR or ORF
entry vectors (see Subheading 3.3.1) and the Gatewaycompatible pK7WGF2 binary vector as follows: mix 1 μL
(50 ng) of entry plasmid (pENTR11-Renilla 3
0 UTR-BS or
pENTR11-Renilla ORF-BS, Fig. 2) with 1 μL (50 ng) of
pK7WGF2. Add 0.5 μL of LR clonase. Incubate the reaction
at room temperature for 1–12 h.
2. Chemically competent TOP10 E. coli cells are transformed
with the recombination reaction. Spread bacteria onto an LB
agar plate containing 100 μg/mL of spectinomycin. Incubate
the plates overnight at 37
C.
3. Screen the appearing colonies by colony PCR. Correct recombination leads to in-frame fusions between the Renilla luciferase and GFP-coding regions. In the resulting plasmids the
miRNA-binding sites are located either in the 3
0 UTR of the
Renilla luciferase-GFP (pK7WGF2-Renilla-3
0 UTR miRNABS) or in the linker region connecting the GFP and Renilla
luciferase domains of the fusion gene (pK7WGF2-RenillaORF miRNA-BS) (Fig. 2).
3.4 Agroinfiltration
To ensure efficient co-delivery of components of the reporter system into plants, agroinfiltration is used. First, the constructed
binary vectors are separately transformed into cells of a suitable
A. tumefaciens strain (e.g., C58C1). Next, the transformed bacterium strains are introduced into N. benthamiana leaves in various
combinations by syringe infiltration.
3.4.1 Agrobacterium
Transformation/Growth
1. Streak C58C1 A. tumefaciens strain from a frozen glycerol
stock onto a YEB-RT plate. Incubate the plate for 3–5 days at
28
C.
2. Inoculate a single colony of C58C1 into 5 mL of YEB medium
containing 20 μg/mL of rifampicin and 5 μg/mL of tetracycline. Shake the culture vigorously (200–250 rpm) overnight at
28
C.
3. Add 2 mL of the overnight culture to 50 mL of YEB medium
(without antibiotics) in a 250 mL flask. Shake the culture
vigorously (250 rpm) at 28
C until it grows to OD 600 of
0.5–1.0 (this generally takes 4–6 h).
4. Chill the culture on ice for 10 min.
5. Centrifuge the cell suspension at 3000 Â g for 10 min at 4
C.
6. Discard supernatant and resuspend the bacterium pellet in
1 mL of ice-cold 20 mM CaCl 2 (see Note 12).
7. Add 1–2 μg of binary plasmid DNA to 100 μL of bacterium
suspension.
8. Freeze mixture in liquid N 2 .
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Ka ´ roly Fa ´ tyol
of Binary 3
0 UTR or ORF
Sensors
1. Set up LR recombination reactions from the 3
0 UTR or ORF
entry vectors (see Subheading 3.3.1) and the Gatewaycompatible pK7WGF2 binary vector as follows: mix 1 μL
(50 ng) of entry plasmid (pENTR11-Renilla 3
0 UTR-BS or
pENTR11-Renilla ORF-BS, Fig. 2) with 1 μL (50 ng) of
pK7WGF2. Add 0.5 μL of LR clonase. Incubate the reaction
at room temperature for 1–12 h.
2. Chemically competent TOP10 E. coli cells are transformed
with the recombination reaction. Spread bacteria onto an LB
agar plate containing 100 μg/mL of spectinomycin. Incubate
the plates overnight at 37
C.
3. Screen the appearing colonies by colony PCR. Correct recombination leads to in-frame fusions between the Renilla luciferase and GFP-coding regions. In the resulting plasmids the
miRNA-binding sites are located either in the 3
0 UTR of the
Renilla luciferase-GFP (pK7WGF2-Renilla-3
0 UTR miRNABS) or in the linker region connecting the GFP and Renilla
luciferase domains of the fusion gene (pK7WGF2-RenillaORF miRNA-BS) (Fig. 2).
3.4 Agroinfiltration
To ensure efficient co-delivery of components of the reporter system into plants, agroinfiltration is used. First, the constructed
binary vectors are separately transformed into cells of a suitable
A. tumefaciens strain (e.g., C58C1). Next, the transformed bacterium strains are introduced into N. benthamiana leaves in various
combinations by syringe infiltration.
3.4.1 Agrobacterium
Transformation/Growth
1. Streak C58C1 A. tumefaciens strain from a frozen glycerol
stock onto a YEB-RT plate. Incubate the plate for 3–5 days at
28
C.
2. Inoculate a single colony of C58C1 into 5 mL of YEB medium
containing 20 μg/mL of rifampicin and 5 μg/mL of tetracycline. Shake the culture vigorously (200–250 rpm) overnight at
28
C.
3. Add 2 mL of the overnight culture to 50 mL of YEB medium
(without antibiotics) in a 250 mL flask. Shake the culture
vigorously (250 rpm) at 28
C until it grows to OD 600 of
0.5–1.0 (this generally takes 4–6 h).
4. Chill the culture on ice for 10 min.
5. Centrifuge the cell suspension at 3000 Â g for 10 min at 4
C.
6. Discard supernatant and resuspend the bacterium pellet in
1 mL of ice-cold 20 mM CaCl 2 (see Note 12).
7. Add 1–2 μg of binary plasmid DNA to 100 μL of bacterium
suspension.
8. Freeze mixture in liquid N 2 .
442
Ka ´ roly Fa ´ tyol