Nicotiana Benthamiana Agroinfiltration

Nicotiana benthamiana agroinfiltration

-  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

-  Four days after infiltration (more or less 96 hours), excise one disc (0.8 cm in diameter, approximately 18-19mg) of each agroinfiltrated leaf.

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 Microplate Luminometer (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 Microplate Luminometer (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 that has to be expressed in a different plant on the same experiment.