9. Thaw the bacteria at 37
C for 5 min.
10. Add 1 mL of YEB medium to the bacteria and incubate the
mixture at 28
C for 2–4 h. This period allows the bacteria to
express the antibiotic resistance genes.
11. Centrifuge bacterium suspension in a microcentrifuge for
1 min at maximum speed.
12. Remove majority of supernatant leaving ~100 μL of YEB
medium on the bacterium pellet.
13. Resuspend bacteria and spread suspension onto a YEB plate
containing appropriate antibiotics (see Note 13).
14. Incubate the plate for 3–5 days at 28
C.
3.4.2 Preparation
of Agrobacteria
for Infiltration
1. Inoculate a single colony of each C58C1 strain of the reporter
system into 5 mL of YEB medium containing appropriate
antibiotics (AGO expression strain, 50 μg/mL kanamycin;
miRNA expression strain, 100 μg/mL spectinomycin; wildtype sensor/mutant sensor strains, 100 μg/mL spectinomycin). Empty expression vector (pK7WG2D and pBIN61) containing bacterium strains should be grown as well, as negative
controls. In addition to the antibiotics, supplement the growth
media with MES and acetosyringone to the final concentrations of 10 mM and 20 μM, respectively.
2. Shake the cultures (250 rpm) at 28
C overnight to reach
saturation.
3. Centrifuge cultures at 3000 Â g for 10 min at 25
C.
4. Decant supernatants and resuspend bacterium pellets in 1 mL
of freshly prepared infiltration medium. Incubate suspensions
at room temperature for at least 3 h.
5. Measure optical density of bacterium suspensions. Prepare
OD 600 ¼ 1 dilutions from suspensions using infiltration
medium.
6. Prepare infiltration mixtures from the strains of the reporter
system (AGO expression strain, miRNA expression strain, sensor/mutant sensor strains). The mixing ratios of the strains have
to be optimized for each AGO/miRNA/sensor combination.
In addition, mixtures containing the appropriate empty vector
strains, instead of the AGO or miRNA expression strains,
should also be prepared as negative controls. In our hands,
mixing the three strains at 1:1:1 ratio works well for most of
the time. Setup of a typical experiment is given in Fig. 1b.
3.4.3 Agroinfiltration
1. N. benthamiana plants are grown at 25
C under long-day
conditions (16-h light, 8-h dark). Four–five-week-old plants
are most suitable for infiltration. The day before the experiment, irrigate plants well in order to ensure that their stomata
are fully open at the time of the infiltration.
An In Vivo Reporter System for RNA Silencing
443
C for 5 min.
10. Add 1 mL of YEB medium to the bacteria and incubate the
mixture at 28
C for 2–4 h. This period allows the bacteria to
express the antibiotic resistance genes.
11. Centrifuge bacterium suspension in a microcentrifuge for
1 min at maximum speed.
12. Remove majority of supernatant leaving ~100 μL of YEB
medium on the bacterium pellet.
13. Resuspend bacteria and spread suspension onto a YEB plate
containing appropriate antibiotics (see Note 13).
14. Incubate the plate for 3–5 days at 28
C.
3.4.2 Preparation
of Agrobacteria
for Infiltration
1. Inoculate a single colony of each C58C1 strain of the reporter
system into 5 mL of YEB medium containing appropriate
antibiotics (AGO expression strain, 50 μg/mL kanamycin;
miRNA expression strain, 100 μg/mL spectinomycin; wildtype sensor/mutant sensor strains, 100 μg/mL spectinomycin). Empty expression vector (pK7WG2D and pBIN61) containing bacterium strains should be grown as well, as negative
controls. In addition to the antibiotics, supplement the growth
media with MES and acetosyringone to the final concentrations of 10 mM and 20 μM, respectively.
2. Shake the cultures (250 rpm) at 28
C overnight to reach
saturation.
3. Centrifuge cultures at 3000 Â g for 10 min at 25
C.
4. Decant supernatants and resuspend bacterium pellets in 1 mL
of freshly prepared infiltration medium. Incubate suspensions
at room temperature for at least 3 h.
5. Measure optical density of bacterium suspensions. Prepare
OD 600 ¼ 1 dilutions from suspensions using infiltration
medium.
6. Prepare infiltration mixtures from the strains of the reporter
system (AGO expression strain, miRNA expression strain, sensor/mutant sensor strains). The mixing ratios of the strains have
to be optimized for each AGO/miRNA/sensor combination.
In addition, mixtures containing the appropriate empty vector
strains, instead of the AGO or miRNA expression strains,
should also be prepared as negative controls. In our hands,
mixing the three strains at 1:1:1 ratio works well for most of
the time. Setup of a typical experiment is given in Fig. 1b.
3.4.3 Agroinfiltration
1. N. benthamiana plants are grown at 25
C under long-day
conditions (16-h light, 8-h dark). Four–five-week-old plants
are most suitable for infiltration. The day before the experiment, irrigate plants well in order to ensure that their stomata
are fully open at the time of the infiltration.
An In Vivo Reporter System for RNA Silencing
443