3. Methods 3.1 Preparing plant material Generation of transgenic Arabidopsis plants (see Note 2, 3). Sterilize the seeds. Sow seeds on MS 1% sucrose agar plates and keep in a cold room in the dark for 2-3 days. Grow them for 7 days in a growth room (our conditions: illumination of µmol/m2sec, 16h light/8h dark cycle, temperature of 22-23°C) and then transfer the seedlings into 6-well plates containing 5 mL MS medium with 1% sucrose in each well (5 seedlings per well). After transfer, grow for 7-10 days more and collect samples. Prepare 5 plates per treatment (10-15 g fresh weight) for large scale IP or IP for the identification of phosphorylation sites. Use 1 plate per treatment (2-3 g fresh weight) for small scale IP. Ligand treatment Dilute PAMP stock solution in distilled water (1 µM for flg22 or elf18). Prepare two different sizes of beakers (for example, 300 mL and 200 mL beakers). Put all Arabidopsis seedlings (after removal of MS media using paper towels) into a bigger beaker. Add PAMP solution or water for mock treatment in the bigger beaker with seedlings and put the small beaker on top of it. Make sure all seedlings are under the water (see Note 4). Put the beaker sandwich into a vacuum chamber and apply vacuum for 2 min. Release the pressure slowly and start counting the time. You can see that the PAMP solution gets into the plants as the colour of the leaves becomes darker. If some parts are not infiltrated, repeat the vacuum infiltration process. After 5 min (or 10-15 min depending on your purpose), discard the PAMP solution. Put the seedlings on paper towels and dry them. Immediately, freeze them in liquid nitrogen and keep samples at -80 C.
3. Methods 3.1 Preparing plant material Generation of transgenic Arabidopsis plants (see Note 2, 3). Sterilize the seeds. Sow seeds on MS 1% sucrose agar plates and keep in a cold room in the dark for 2-3 days. Grow them for 7 days in a growth room (our conditions: illumination of µmol/m2sec, 16h light/8h dark cycle, temperature of 22-23°C) and then transfer the seedlings into 6-well plates containing 5 mL MS medium with 1% sucrose in each well (5 seedlings per well). After transfer, grow for 7-10 days more and collect samples. Prepare 5 plates per treatment (10-15 g fresh weight) for large scale IP or IP for the identification of phosphorylation sites. Use 1 plate per treatment (2-3 g fresh weight) for small scale IP. Ligand treatment Dilute PAMP stock solution in distilled water (1 µM for flg22 or elf18). Prepare two different sizes of beakers (for example, 300 mL and 200 mL beakers). Put all Arabidopsis seedlings (after removal of MS media using paper towels) into a bigger beaker. Add PAMP solution or water for mock treatment in the bigger beaker with seedlings and put the small beaker on top of it. Make sure all seedlings are under the water (see Note 4). Put the beaker sandwich into a vacuum chamber and apply vacuum for 2 min. Release the pressure slowly and start counting the time. You can see that the PAMP solution gets into the plants as the colour of the leaves becomes darker. If some parts are not infiltrated, repeat the vacuum infiltration process. After 5 min (or 10-15 min depending on your purpose), discard the PAMP solution. Put the seedlings on paper towels and dry them. Immediately, freeze them in liquid nitrogen and keep samples at -80 C.