(h) M13rev: 5
0 -caggaaacagctatgac
(i) MP-GV1rev: 5
0 -cagagaaagcggacagaaaaccgctgacatcttcac
(j) mRFPfw: 5
0 -gcctcctccgaggacgtc
2.2 Transient Gene
Expression
1. A. tumefaciens strain GV3101 [65] competent for electrotransformation.
2. pB7-NLS-MCP:GFP, for the expression of MCP fused to
eGFP and targeted to the nucleus [35].
3. Incubator for agar plates at 28
C.
4. Shaker for liquid cultures (50 mL falcon tubes) for incubation
at 180–200 rpm and 28
C.
5. Sterile 50 mL falcon tubes.
6. Antibiotics, 1000Â stock solutions, sterilized by filtration
through 0.22 μm pore-size membrane:
(a) Rifampicin: 50 mg/mL (in DMSO).
(b) Gentamicin: 50 mg/mL (in water).
7. Spectrophotometer (to measure absorbance at 600 nm).
8. Centrifuge (compatible with 50 mL falcon tubes and
5000 Â g).
9. Sterile distilled water.
10. Greenhouse or growth chamber for N. benthamiana plants
(16 h/8 h day/night and 24
C/22
C day/night
temperature).
11. N. benthamiana plants, 4–5 weeks old.
12. 1.0–2.5 mL Syringes without needle.
13. Hypodermic needles (e.g., blue or orange needles,
0.5–0.6 mm diameter, respectively).
2.3 Fluorescence
Imaging
1. Epifluorescence, spinning-disk, or laser scanning confocal
microscope equipped with (1) specific filtering for GFP and
RFP fluorescence; (2) low-magnification objective (e.g., 10Â
or 20Â) and high-magnification, high-optical-numerical-aperture objective (e.g., 60Â or 100Â); and (3) a detection system
compatible with high-frame-rate time-lapse image acquisition.
2. Microscopy glass slides and coverslips (thickness compatible
with available objectives).
3. Scotch tape (e.g., Magic™ Tape 1/2
00 , 3 M).
4. Cork borer (0.7–1.0 cm diameter, for leaf disk preparation).
5. Vacuum pump with vacuum desiccator.
6. Sterile distilled water.
7. Microscope image acquisition and analysis software (e.g., ImageJ https://imagej.nih.gov/ij/index.html).
110
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein
0 -caggaaacagctatgac
(i) MP-GV1rev: 5
0 -cagagaaagcggacagaaaaccgctgacatcttcac
(j) mRFPfw: 5
0 -gcctcctccgaggacgtc
2.2 Transient Gene
Expression
1. A. tumefaciens strain GV3101 [65] competent for electrotransformation.
2. pB7-NLS-MCP:GFP, for the expression of MCP fused to
eGFP and targeted to the nucleus [35].
3. Incubator for agar plates at 28
C.
4. Shaker for liquid cultures (50 mL falcon tubes) for incubation
at 180–200 rpm and 28
C.
5. Sterile 50 mL falcon tubes.
6. Antibiotics, 1000Â stock solutions, sterilized by filtration
through 0.22 μm pore-size membrane:
(a) Rifampicin: 50 mg/mL (in DMSO).
(b) Gentamicin: 50 mg/mL (in water).
7. Spectrophotometer (to measure absorbance at 600 nm).
8. Centrifuge (compatible with 50 mL falcon tubes and
5000 Â g).
9. Sterile distilled water.
10. Greenhouse or growth chamber for N. benthamiana plants
(16 h/8 h day/night and 24
C/22
C day/night
temperature).
11. N. benthamiana plants, 4–5 weeks old.
12. 1.0–2.5 mL Syringes without needle.
13. Hypodermic needles (e.g., blue or orange needles,
0.5–0.6 mm diameter, respectively).
2.3 Fluorescence
Imaging
1. Epifluorescence, spinning-disk, or laser scanning confocal
microscope equipped with (1) specific filtering for GFP and
RFP fluorescence; (2) low-magnification objective (e.g., 10Â
or 20Â) and high-magnification, high-optical-numerical-aperture objective (e.g., 60Â or 100Â); and (3) a detection system
compatible with high-frame-rate time-lapse image acquisition.
2. Microscopy glass slides and coverslips (thickness compatible
with available objectives).
3. Scotch tape (e.g., Magic™ Tape 1/2
00 , 3 M).
4. Cork borer (0.7–1.0 cm diameter, for leaf disk preparation).
5. Vacuum pump with vacuum desiccator.
6. Sterile distilled water.
7. Microscope image acquisition and analysis software (e.g., ImageJ https://imagej.nih.gov/ij/index.html).
110
Eduardo Jose ´ Pen ˜ a and Manfred Heinlein