13. Purify pDONR221.Csy4∗ plasmid DNA from each culture
using a plasmid miniprep kit according to the manufacturer’s
instructions.
14. Verify the removal of the NLS by DNA sequencing with primer
DONRrev.
15. Recombine the following constructs in two separate Gateway
LR Clonase II reactions according to the manufacturer’s
instructions:
(a) pDONR221.Csy4∗ and pGWB405 to make pGWB405.
Csy4∗-GFP.
(b) pDONR221.Csy4∗-KpnI-NLS-KpnI and pGWB405 to
make pGWB405.Csy4∗-NLS-GFP.
16. Electroporate 1 μL of each LR recombination reaction into
50 μL aliquots of XL1 Blue MRF’ cells, and then incubate cells
in 1 mL LB medium without antibiotics at 37
C for 1 h.
17. Plate 100 μL of each transformation on LB-agar plates containing 100 μg/mL spectinomycin, and incubate overnight at
37
C.
18. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 100 μg/mL spectinomycin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
19. Purify pGWB405.Csy4∗-GFP and pGWB405.Csy4∗-NLS-GFP plasmids from each culture using a plasmid miniprep
kit according to the manufacturer’s instructions.
20. Verify the presence of the Csy4∗ inserts by DNA sequencing
with primers attB1 adapter and attB2 adapter.
21. Electroporate 0.5 μL of a verified pGWB405.Csy4∗-GFP and
pGWB405.Csy4∗-NLS-GFP plasmid, respectively, into 50 μL
aliquots of A. tumefaciens AGL1 cells each, and then incubate
cells in 1 mL LB medium without antibiotics at 28
C for 2 h.
22. Plate 10–25 μL of each transformation on LB-agar plates containing 100 μg/mL spectinomycin and 50 μg/mL rifampicin,
and incubate at 28
C for 2–3 days.
23. Inoculate separate 5 mL aliquots of LB liquid medium containing 100 μg/mL spectinomycin and 50 μg/mL rifampicin with
one well-separated colony from each plate, and incubate at
28
C with shaking for 2 days.
24. Prepare a glycerol stock from each culture by mixing 750 μL
culture with 750 μL sterile glycerol and shock-freezing in
liquid nitrogen. Store the glycerol stocks at À80
C.
3.2 Tagging of the
PVX Genome
1. Digest 25 μL of each of the plasmids pTRA.PVX.mCherry-2ACP and pMA-RQ.NheI-2xcsy-NheI with NheI in separate
RNA Imaging with Csy4
167
using a plasmid miniprep kit according to the manufacturer’s
instructions.
14. Verify the removal of the NLS by DNA sequencing with primer
DONRrev.
15. Recombine the following constructs in two separate Gateway
LR Clonase II reactions according to the manufacturer’s
instructions:
(a) pDONR221.Csy4∗ and pGWB405 to make pGWB405.
Csy4∗-GFP.
(b) pDONR221.Csy4∗-KpnI-NLS-KpnI and pGWB405 to
make pGWB405.Csy4∗-NLS-GFP.
16. Electroporate 1 μL of each LR recombination reaction into
50 μL aliquots of XL1 Blue MRF’ cells, and then incubate cells
in 1 mL LB medium without antibiotics at 37
C for 1 h.
17. Plate 100 μL of each transformation on LB-agar plates containing 100 μg/mL spectinomycin, and incubate overnight at
37
C.
18. The next day, inoculate several 5 mL aliquots of LB liquid
medium containing 100 μg/mL spectinomycin with a wellseparated colony each, and incubate overnight at 37
C with
shaking.
19. Purify pGWB405.Csy4∗-GFP and pGWB405.Csy4∗-NLS-GFP plasmids from each culture using a plasmid miniprep
kit according to the manufacturer’s instructions.
20. Verify the presence of the Csy4∗ inserts by DNA sequencing
with primers attB1 adapter and attB2 adapter.
21. Electroporate 0.5 μL of a verified pGWB405.Csy4∗-GFP and
pGWB405.Csy4∗-NLS-GFP plasmid, respectively, into 50 μL
aliquots of A. tumefaciens AGL1 cells each, and then incubate
cells in 1 mL LB medium without antibiotics at 28
C for 2 h.
22. Plate 10–25 μL of each transformation on LB-agar plates containing 100 μg/mL spectinomycin and 50 μg/mL rifampicin,
and incubate at 28
C for 2–3 days.
23. Inoculate separate 5 mL aliquots of LB liquid medium containing 100 μg/mL spectinomycin and 50 μg/mL rifampicin with
one well-separated colony from each plate, and incubate at
28
C with shaking for 2 days.
24. Prepare a glycerol stock from each culture by mixing 750 μL
culture with 750 μL sterile glycerol and shock-freezing in
liquid nitrogen. Store the glycerol stocks at À80
C.
3.2 Tagging of the
PVX Genome
1. Digest 25 μL of each of the plasmids pTRA.PVX.mCherry-2ACP and pMA-RQ.NheI-2xcsy-NheI with NheI in separate
RNA Imaging with Csy4
167