Measurement of luciferase activity in tobacco

Dual NanoLuc assay in tobacco

This is a slightly updated protocol for "Measurement of luciferase activity in tobacco" from Plant Biotechnology 38, 89–99 (2021) DOI: 10.5511/plantbiotechnology.20.1209a.

Plant material

N. benthamiana plants at 3-6 week after germination are used for infiltration.

Binary vector 

We use pGWB vectors to express luciferase genes. Usually, NanoLuc and Fluc are used for reporter and internal reference gene, respectively. For example, promoter-X-NanoLuc and CaMV35S promoter-Fluc.  

Agrobacterium 

Agrobacterium strain EHA101, EHA105, and MP90 work well.

1) Inoculate a small piece of glycerol stock of Agrobacterium (stored at -80℃) into 2 mL LB medium supplemented with appropriate antibiotics (in case of pGWB602, 100mg/L of spectinomycin) with 200rpm shaking at 28°C overnight. If you need to boost the expression in tobacco, prepare Agrobacterium culture with p19 vector (in case of pBIC p19, 50mg/L of kanamycin). 

2) On the next day, measure OD₆₀₀ of the Agrobacterium culture (measure x10 dilution). Add Agrobacterium suspension into 10mL LB (in a 50mL tube) to adjust the final OD₆₀₀ to 0.25. Antibiotics is not necessary to add. Start culture with 200rpm shaking at 28°C for 3-4hr. OD₆₀₀ should not exceed 1.0. 

Agro infiltration

Agro infiltration buffer (make just before use)

Acetosyringone (4'-Hydroxy-3',5'-dimethoxyacetophenone) stock solution (150mM) = 29.4 mg/mL ethanol, stored at -20°C

3) At OD₆₀₀=0.5~1.0, stop culture. Centrifuge the suspension at 8,000rpm for 10min at room temperature.

4) Remove supernatant and suspend the bacterial pellet into 10mL of Agro infiltration buffer. 

5) Centrifuge at 8,000rpm for 10min at room temp.

6) Remove supernatant

7) Centrifuge at 8,000rpm for 1min at room temp and remove the trace amount of supernatant completely with pipette.

8) Suspend the pellet to 10mL of Agro infiltration buffer. If necessary, adjust OD_600. We usually adjust the concentration at OD₆₀₀=0.6. If you plan to use p19, mix p19 Agro suspension (usually 1:1). 

9) Infiltrate Agro suspension into the backside of tobacco leaf with 1mL syringe without needle. 

10) After infiltration, wipe off the extra Agro suspension on the leaf with paper towel and mark the infiltrated areas with an oily marking pen.

Measurement of NanoLuc activity

To detect the luciferase activity, we use a commercial kit, Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega, hereinafter referred to as “Nano-Glo kit”) and Passive Lysis 5X Buffer (Promega, hereinafter referred to as “5X PLB”) according to the manufacturer’s instructions. To measure the activity, we use TriStar LB941 (Berthold Technologies) or GloMax explorer (Promega), according to the manufacturer’s instructions. For economical use of Nano-Glo kit, we do not use auto-injector but add reagents manually. 

11) Prepare reagents and buffer. Dilute 5X PLB to 1X with deionized water just before use. Prepare ONE-Glo EX Luciferase Assay reagent (hereinafter referred to as “EX”) and NanoDLR Stop & Glo reagent (hereinafter referred to as “SG”), components of the Nano-Glo kit, according to the manufacturer’s instructions. EX can be stored at -20℃ and SG should be made just before use. 

12) After 3-5 day post infiltration, cut out a small piece of the infiltrated tobacco leaf (usually the size of 5mm x 5mm) and manually grind it in a 1.5mL tube with a plastic pestle. After it is completely homogenized, add 100uL of 1X PLB and mix well. 

13) Set up the luminometer according to the manufacturer’s instructions. For GloMax explorer, we use a preinstalled protocol, luminescence light plate, to measure the luminescence. Usually, one second integration (=detection time) is enough. If the signal intensity is lower than 10,000 unit, extend the integration time (10s-30s) or increase the sample amount for measurement. 

14) Load 5ul of the leaf extract onto a well of a 96 well titer plate, add 5ul of EX in the well, and mix well by pipetting. 

15) Measure luminescence (=Fluc activity). 

16) Add 5ul of SG in the well and mix well by pipetting.

17) Measure luminescence (=NanoLuc activity). 

18) If NanoLuc and Fluc vectors are co-transfected by Agrobacterium infection, the relative NanoLuc activity is calculated as NanoLuc activity/Fluc activity.