Protocol based on Johansson et al., 2015.
Materials
- Plastic 50 ml centrifuge tubes
- Plastic 15ml falcon tubes
- 5ml pipette tips
- Distilled water
- Plastic bacterial inoculation loops
- Petri dishes
- Corning Costar Flat Bottom 6-well Cell Culture Plates (Fisher)
- Tea strainer
- Conductivity meter: Jenway 3540 pH and conductivity meter
- Conductivity probe: Glass bodied 4mm diameter, 120mm reach probe (Part code 027 815)
- 10μS and 84μS conductivity standard (Part code 025 139 and 025 164 respectively)
Experimental design
- Always take along a positive and a negative control
- Make sure the plants used look good. There is no point in doing the experiment with subpar plants
- Make sure to calibrate the conductivity meter before measuring
Before start
- 24 hour prior to experiment start: Grow Agrobacteria strains from glycerol in appropriate antibiotics. Grow in 28°C over night.
- Day before or just before experiment start: Rinse one 6 well culture plate for each plant line/treatment to be investigated three times in deionized water.
Agro-infiltration
- Measure OD600 of Agrobacterial strains
- Calculate required volume of bacterial culture for a final OD600 = 0.25/construct in the final volume
- Mix strains, spin down at 2000x g, RT
- Decant supernatant and resuspend bacteria in infiltration buffer (10mM MgCl2, 10mM MES-KOH pH5.6, 200μM)
- Leave the mixed cultures at room-temperature in dark for 2 hours prior to infiltration
- Infiltrate 4-5 week old N. benthamiana plants, fully infiltrate 2 leaves while making sure to minimize mechanical damage, mark un-infiltrated zones
Leaf discs
- 1dpi (optimize this for each specific HR system) make leaf discs from infiltrated leafs using a cork borer number 4 (8mm) by punching them out against a piece of Styrofoam. If possible, avoid the midvein and edges of the leaf. Put the discs into a 50 ml plastic centrifuge tube. Approximately 40-50 discs per construct are needed.
- Pour the leaf discs into a tea strainer and rinse under a mild stream of deionized water
- Further wash the discs by transferring them into Petri dishes filled with deionized water for 1hour
- Add 10 ml of ddH20 to each plate well
- Transfer 6 leaf discs to each well of a 6-well cell culture plate filled with 10ml ddH20 (6x6=36 leaf discs total per treatment)
- Measure conductivity:
- Gently pipet up and down twice to mix
- Pipet 5ml into a 15ml falcon tube making sure not to harm leaf discs
- Calibrate the conductivity meter before use
- After measurements return the liquid into the corresponding well
- The first measurement should be done as soon as possible after transferring the leaf discs, and is the background. Subsequent measurements can be done at the hour scale or over several days (the optimal conditions need to be determined for each HR system)
- In between measurements leave plates with leaf discs mildly rocking on a rocking and waving shaker
- At the end of the experiment collect the leaf discs in the 15ml falcon tubes, add the remaining liquid in the tube (make sure all leaf discs are submerged or in contact with water) and boil for 10min in a 95°C water bath
- Measure the total conductivity
Data
- In the first tab of the excel sheet add the conductivity data. In the second tab which number corresponds to which treatment or construct, and in the third tab the time of every measurement. The first measurement starts at 0
- Use the R script to make graphs!