5. Dilute the Agrobacterium suspensions 1:10 in infiltration
medium and measure their optical density at 600 nm using a
spectrophotometer.
6. Rest cells for 1 h in the dark at room temperature.
7. Adjust the optical density of each (undiluted) Agrobacterium
suspension to 0.2 using infiltration buffer, and then mix a strain
transformed with a Csy4∗ reporter and the strain carrying p19
1:1, so that the final optical density for each individual AGL1
strain is 0.1.
8. Gently turn a leaf previously inoculated with tagged PVX
(Subheading 3.3) to expose the underside, support with your
hand, and with extreme care use the 25G needle to pierce only
the lower epidermis of the leaf in several spots about 1–2 cm
apart (see Note 10).
9. Fill a 1 mL syringe (without a needle) with the Agrobacterium
suspension and while supporting the leaf from the opposite
side, press the syringe mouth firmly against one of the incisions
and depress the plunger of the syringe; the leaf tissue will
change color as the air space is filled with the Agrobacterium
suspension (see Note 11).
10. Repeat until the entire leaf area has been infiltrated. This
requires about 1–2 mL suspension.
11. Label the plant and return to the growth facility for 3–5 days
before imaging.
3.5 Visualization of
Tagged PVX vRNA in
N. benthamiana Leaf
Epidermal Cells
1. Imaging should be performed 3–4 days after agro-infiltration
(Subheading 3.4), and 4–6 days after PVX inoculation (Subheading 3.3).
2. Detach a virus-inoculated and agro-infiltrated leaf from a plant
and attach it to a glass slide using double-sided tape with the
lower epidermis facing upwards.
3. Image epidermal cells on an upright confocal laser scanning
microscope. A 10Â long-distance air lens is suitable for identifying mCherry-expressing infection sites. For imaging of subcellular vRNA localization, a 40Â water-dipping lens is
required, with a drop of water placed directly onto the leaf
epidermis. Use sequential imaging for optimal channel separation. GFP is excited at 488 nm and detected at 495–525 nm;
mCherry is excited at 594 nm and detected at 600–630 nm (see
Note 12).
3.6 Construction of
Csy4∗ Expression
Plasmid for RNA
Imaging in N. crassa
1. Amplify the open reading frames of Csy4∗ from pDONR221.
Csy4∗ (Subheading 3.1) and mBasicGFP from pLS3 by PCR
with primer pairs Csy4-IF-F/Csy4-IF-R and GFP-IF-F/GFPIF-R, respectively. Use HiFi DNA polymerase according to the
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medium and measure their optical density at 600 nm using a
spectrophotometer.
6. Rest cells for 1 h in the dark at room temperature.
7. Adjust the optical density of each (undiluted) Agrobacterium
suspension to 0.2 using infiltration buffer, and then mix a strain
transformed with a Csy4∗ reporter and the strain carrying p19
1:1, so that the final optical density for each individual AGL1
strain is 0.1.
8. Gently turn a leaf previously inoculated with tagged PVX
(Subheading 3.3) to expose the underside, support with your
hand, and with extreme care use the 25G needle to pierce only
the lower epidermis of the leaf in several spots about 1–2 cm
apart (see Note 10).
9. Fill a 1 mL syringe (without a needle) with the Agrobacterium
suspension and while supporting the leaf from the opposite
side, press the syringe mouth firmly against one of the incisions
and depress the plunger of the syringe; the leaf tissue will
change color as the air space is filled with the Agrobacterium
suspension (see Note 11).
10. Repeat until the entire leaf area has been infiltrated. This
requires about 1–2 mL suspension.
11. Label the plant and return to the growth facility for 3–5 days
before imaging.
3.5 Visualization of
Tagged PVX vRNA in
N. benthamiana Leaf
Epidermal Cells
1. Imaging should be performed 3–4 days after agro-infiltration
(Subheading 3.4), and 4–6 days after PVX inoculation (Subheading 3.3).
2. Detach a virus-inoculated and agro-infiltrated leaf from a plant
and attach it to a glass slide using double-sided tape with the
lower epidermis facing upwards.
3. Image epidermal cells on an upright confocal laser scanning
microscope. A 10Â long-distance air lens is suitable for identifying mCherry-expressing infection sites. For imaging of subcellular vRNA localization, a 40Â water-dipping lens is
required, with a drop of water placed directly onto the leaf
epidermis. Use sequential imaging for optimal channel separation. GFP is excited at 488 nm and detected at 495–525 nm;
mCherry is excited at 594 nm and detected at 600–630 nm (see
Note 12).
3.6 Construction of
Csy4∗ Expression
Plasmid for RNA
Imaging in N. crassa
1. Amplify the open reading frames of Csy4∗ from pDONR221.
Csy4∗ (Subheading 3.1) and mBasicGFP from pLS3 by PCR
with primer pairs Csy4-IF-F/Csy4-IF-R and GFP-IF-F/GFPIF-R, respectively. Use HiFi DNA polymerase according to the
170
David Burnett et al.