Supplemental Information
Experimental procedures
In-gel MAP kinase assay
Seedlings were grown for 7 days on solid MS medium and then transferred to wells of 24
well microtiter plates (Nunc) containing in each well 500μl MS liquid medium and grown
for 10 more days. MS liquid medium was then replenished to 500µl and after 2 h flg22 was
added to a final concentration of 100nM. After 0, 5, 10 and 30minutes aerial tissue was
transferred to 2ml eppendorf microcentrifuge tubes, frozen in liquid nitrogen and
homogenized. Protein extraction and protein kinase activity assays were performed as
described (Nühse et al., 2000). Kinase activity was visualized by phosphoimaging (Typhoon
8600 Phosphor Imager and Image Eraser, Molecular Dynamics).
Ethylene Measurements
Samples were taken from leaves of 4-week-old soil-grown plants using a cork borer (ø
0,6cm) to excise 24 leaf discs from at least 12 independent plants. Leaf discs were floated
over night on dH2O before transferring 3x8 leaf discs per genotype and treatment into
hermetic vials containing either 1ml dH2O or 1ml dH2O with 100nM flg22. Vials were
sealed with rubber septa and incubated on a shaker for 4 h at room temperature. After 4 h
ethylene production was measured by gas chromatography (GC). The analysis was
performed on an Agilent 6890 GC connected to an Agilent 5975N mass selective detector
(MSD, Agilent) operating in split mode with a ratio of 10 to 1. 100µl of the gas phase were
taken from the hermetically closed vial with a gas-tight syringe and injected in the GC.
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Ethylene was separated on an Agilent GS-GasPro column (60m, ø 0,32mm) at 90°C and
1.4ml/min. The MSD was run in the ”selected ion monitoring” (SIM) mode, measuring
Ethylene fragment ions of 24,1; 25,1; 26,1 and 27,1 amu. Additional to the fragment masses,
identification of the ethylene peak was based on the retention time of an ethylene standard
(Fluka, Deisenhofen, Germany) run under same conditions and the ratio of ion abundances.
To quantify ethylene, the area sum of all four ion counts was integrated using Chemstation
software (Agilent). For more precise analysis, integrals of the void volume and the ethylene
peaks were calculated and their ratio to each other was determined. For each genotype and
treatment, average was calculated from three replicates. The obtained value was then used to
express relative ethylene quantities in the different samples.
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