TITLE: Agrobacterium-Mediated Transient Expression in Nicotiana benthamiana Leaves
AUTHORS: Maryam Rahmati Ishka

[DESCRIPTION]
This procedure outlines the Agrobacterium infiltration of tobacco leaves for the transient expression of fluorescently labeled proteins.

[BEFORE_START]
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[GUIDELINES]
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[STEPS]
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
1. Refresh bacteria containing the construct of interest from glycerol stock on agar YEP (or any other bacterial media such as LB) medium with an appropriate antibiotic. Incubate at 28°C for two days. If A. tumefaciens GV3101 is used add rifampicin.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
2. Inoculate 5 mL of liquid YEP medium with appropriate antibiotic in a test tube. Grow at 28°C for 24 hours
with shaking at 250-300 rpm. Be sure to grow bacterial strain with p19 as a silencing inhibitor as well!
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
3. Measure the OD600 using 100 µL of bacterial culture.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
4. Calculate the culture volume needed for an OD600 of 0.2 - 0.5 per construct for a 4 mL total volume of Infiltration medium. 4 mL are used for test spot infiltration. The specific OD600 to use depends on how
well expressed your construct is in N. benth, the general range is 0.1 – 0.5. Use an OD600 of 0.3 for P19.
(I use 0.3 for all constructs and it works well).
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
5. Pipette the appropriate volume of each strain together in 2 mL tubes.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
6. Centrifuge combined bacteria at 8000×g for 2 min at room temperature. Carefully remove all supernatant.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
7. Add 200 mM acetosyringone to the Infiltration medium at a 1:1000 ratio.
Note: acetosyringone is light-sensitive, keep out of light until ready for use.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
8. Add 2 mL of infiltration medium with acetosyringone to each pellet, and resuspend. Leave in the dark at room temperature for 1–2 hrs (or even longer but not overnight).  
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
9. Infiltrate 4-week-old N. benthamiana (pre-flowering) leaf using 1 mL syringe.
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
10. Grow plants for 2-3 days after infiltration (up to 5 days, depending on expression level of constructs).
SECTION: Transform constructs into Agrobacterium tumefaciens strains that are compatible with vectors. Strain GV3101 is typically used (GV2260 can also be used). Make glycerol stock of.
11. Punch 1 cm leaf discs from leaves and observe under the microscope to visualize the fluorescence.