Jul 17, 2024

Public workspaceAgrobacterium-Mediated Transient Expression in Nicotiana benthamiana Leaves

DOI
dx.doi.org/10.17504/protocols.io.rm7vzj7prlx1/v1
  • 1Boyce Thompson Institute (BTI)
Maryam Rahmati Ishka
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzj7prlx1/v1
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Protocol Citation: Maryam Rahmati Ishka 2024. Agrobacterium-Mediated Transient Expression in Nicotiana benthamiana Leaves. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj7prlx1/v1
Manuscript citation:
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License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 17, 2024
Last Modified: July 17, 2024
Protocol Integer ID: 103602
Keywords: Agrobacterium infiltration, Nicotiana benthamiana, plant construct delivery, transient protein expression
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Abstract
This procedure outlines the Agrobacterium infiltration of tobacco leaves for the transient expression of fluorescently labeled proteins.
Image Attribution
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Guidelines
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Materials
Infiltration medium: 10 mM MgCl2, 10 mM MES (pH 5.6). Autoclave for 20 min. Add acetosyringone immediately before using it ReagentMgCl2Applied Biosystems (ThermoFisher Scientific) Reagent20X MES BufferThermo Fisher ScientificCatalog #NP0002 ReagentAcetosyringoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #2478-38-8 Reagent1g Rifampicin USP GradeG-BiosciencesCatalog #RC-191 to a final concentration of 200 μM.
Safety warnings
Attention
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Ethics statement
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Before start
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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.
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.
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.
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!
Measure the OD600 using 100 µL of bacterial culture.
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).
Pipette the appropriate volume of each strain together in 2 mL tubes.
Centrifuge combined bacteria at 8000×g for 2 min at room temperature. Carefully remove all supernatant.
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.
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).  
Infiltrate 4-week-old N. benthamiana (pre-flowering) leaf using 1 mL syringe.
Grow plants for 2-3 days after infiltration (up to 5 days, depending on expression level of constructs).
Punch 1 cm leaf discs from leaves and observe under the microscope to visualize the fluorescence.
Protocol references
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