Nicotianabenthamianaagroinfiltration
-Transfer the GBelements listed as ‘GBelements used in the experiment’ to Agrobacterium tumefaciens strain GV3101 by electroporation.
-Set overnight-grown bacterial cultures for agroinfiltration.
-Pellet and resuspend the cultures on agroinfiltration solution (10mM MES, pH 5.6, 10mM MgCl2, and 200µM acetosyringone) to an optical density of 0.2 at 600nm.
-Incubate the bacterial suspensions for 2 hours at room temperature on a horizontal rolling mixer.
-Mix the bacterial suspensions in equal volumes.
-Agroinfiltrate the mix on the three youngest leaves of each plant through the abaxial using a 1ml needle-free syringe.
Sampling
-Three days after infiltration (more or less 72 hours), excise one disc (0.8 cm in diameter, approximately 18-19mg) of each agroinfiltrated leaf.
-Transfer each disc to a 24-well plate with 350µl of dexamethasone at the concentration indicated on the description of the experiment (D1756 Sigma Aldrich resuspended in a small amount of DMSO and diluted to the desired concentration in distilled water).
-Keep the plate at 24ºC/20ºC 16h light/8h darkness and freeze samples in 1.5ml eppendorfs with liquid nitrogen at 24 hours.
Luciferase/Renilla activity determination
-Homogenize leaf discs in 1.5ml eppendorfs using steel balls and the RETSCH ball mill MM 400.
-Extract each sample with 150µl of ‘Passive Lysis Buffer’.
-Vortex it and centrifugue it 15 minutes (14000 x g) at 4ºC.
-Take the supernatant and dilute it 2:3 in Passive Lysis Buffer (24µl of supernatant + 36µl of Passive Lysis Buffer) resulting in the working plant extract.
-For Fluc activity determination use a white 96-well plate. On each well, mix 10µl of working plant extract with 40µl of LARII. Incubate for 10 minutes and measure the luciferase activity using a GloMax 96 MicroplateLuminometer (Promega) with a 2-second delay and a 10-second measurement.
-For Rluc activity determination add 40µl of Stop&Glo Reagent with the renilla substrate to each well of the plate. Incubate for 10 minutes and measure the luciferase activity using a GloMax 96 MicroplateLuminometer (Promega) with a 2-second delay and a 10-second measurement.
Data analysis
-Calculate the Fluc and Rluc mean for each well based on the three measurements.
-Calculate the Fluc/Rluc ratio for each well.
-Express the Fluc/Rluc ratios as RPUs (relative promoter units) by dividing each ratio by the Fluc/Rluc ratio obtained for the reference standard GB0166 at the 24 hours timepoint that was expressed in a different plant on the same experiment.