8. Transplant each seedling to a single pot and cover for 2 days
with cling film or plastic cover. Move the pots into growth
chamber to grow plants for 3–4 weeks at 24
C.
9. Water plants with fertilizer NPK 20-20-20 once a week to
boost the vegetative growth.
3.3 Infiltration
of Nicotiana
Benthamiana
1. Inoculate different Agrobacterium EHA105 strains harboring
tripartite split-GFP-based constructs and the p19 strain each
into 3 mL of LB medium with the corresponding antibiotics by
picking a single big colony on the LB plate. Grow the
pre-culture in a shaker at 180–220 rpm overnight at 28
C.
2. Inoculate 5–10 μL of Agrobacterium pre-culture into 10 mL of
LB medium containing the corresponding antibiotic, 10 mM
MES [pH 5.7] and 20 μM Acetosyringone in a 100-mL Erlenmeyer flask. Incubate the culture in a shaker at 180–220 rpm
for 14–16 h at 28
C.
3. Measure the optical density at 600 nm (OD 600 ) with the
spectrophotometer using a cuvette containing 1 mL of LB
medium as blank.
4. Centrifuge Agrobacterium culture (OD 600 < 1) at 4000 Â g for
15 min at 4
C.
5. Discard supernatant and resuspend Agrobacterium culture in
infiltration medium by gentle pipetting to reach an OD 600
suspension of 0.25–0.5. Let the Agrobacterium culture stand at
room temperature for 2–3 h in the dark.
6. Calculate the amount of Agrobacterium needed for a final
1-mL suspension per leaf using the formula V suspension ¼ V final  OD 600 final /OD 600 suspension . For Agrobacterium
strains harboring tripartite split-GFP-based constructs and the
p19 strain, the optimal OD 600 final ranges from 0.0625 to
0.125 (see Note 3).
7. Mix Agrobacterium culture harboring tripartite split-GFPbased constructs together with the p19 culture with the same
OD 600 suspension in equal proportion to reach the desired
OD 600 final (see Note 3).
8. Select N. benthamiana plants of the same age and size. Keep
the plants in the dark without watering for 16–24 h. Transfer
N. benthamiana plants back to the light and water the plants
1–2 h prior to agroinfiltration.
9. Apply the co-infiltration with 1-mL syringe without needle.
Gently press the syringe to infiltrate the suspension culture
into the abaxial epidermis of the healthy 2
0 and 3
0 leaves of
plants, and exert a counter-pressure with your finger on the
other side. Mark the infiltrated leaf by adhering tape to the
petiole. Return infiltrated plants to growth chamber and grow
for another 2–5 days (see Note 4).
Tripartite Split-sfGFP Assay
331
with cling film or plastic cover. Move the pots into growth
chamber to grow plants for 3–4 weeks at 24
C.
9. Water plants with fertilizer NPK 20-20-20 once a week to
boost the vegetative growth.
3.3 Infiltration
of Nicotiana
Benthamiana
1. Inoculate different Agrobacterium EHA105 strains harboring
tripartite split-GFP-based constructs and the p19 strain each
into 3 mL of LB medium with the corresponding antibiotics by
picking a single big colony on the LB plate. Grow the
pre-culture in a shaker at 180–220 rpm overnight at 28
C.
2. Inoculate 5–10 μL of Agrobacterium pre-culture into 10 mL of
LB medium containing the corresponding antibiotic, 10 mM
MES [pH 5.7] and 20 μM Acetosyringone in a 100-mL Erlenmeyer flask. Incubate the culture in a shaker at 180–220 rpm
for 14–16 h at 28
C.
3. Measure the optical density at 600 nm (OD 600 ) with the
spectrophotometer using a cuvette containing 1 mL of LB
medium as blank.
4. Centrifuge Agrobacterium culture (OD 600 < 1) at 4000 Â g for
15 min at 4
C.
5. Discard supernatant and resuspend Agrobacterium culture in
infiltration medium by gentle pipetting to reach an OD 600
suspension of 0.25–0.5. Let the Agrobacterium culture stand at
room temperature for 2–3 h in the dark.
6. Calculate the amount of Agrobacterium needed for a final
1-mL suspension per leaf using the formula V suspension ¼ V final  OD 600 final /OD 600 suspension . For Agrobacterium
strains harboring tripartite split-GFP-based constructs and the
p19 strain, the optimal OD 600 final ranges from 0.0625 to
0.125 (see Note 3).
7. Mix Agrobacterium culture harboring tripartite split-GFPbased constructs together with the p19 culture with the same
OD 600 suspension in equal proportion to reach the desired
OD 600 final (see Note 3).
8. Select N. benthamiana plants of the same age and size. Keep
the plants in the dark without watering for 16–24 h. Transfer
N. benthamiana plants back to the light and water the plants
1–2 h prior to agroinfiltration.
9. Apply the co-infiltration with 1-mL syringe without needle.
Gently press the syringe to infiltrate the suspension culture
into the abaxial epidermis of the healthy 2
0 and 3
0 leaves of
plants, and exert a counter-pressure with your finger on the
other side. Mark the infiltrated leaf by adhering tape to the
petiole. Return infiltrated plants to growth chamber and grow
for another 2–5 days (see Note 4).
Tripartite Split-sfGFP Assay
331