Extraction and Purification of DSF Signaling Molecules

The protocol for extraction and purification of DSF family components was described previously48 (link). In brief, Xcc strains were cultured in liquid medium for 24–48 hours and 50 mL of bacterial supernatant was collected by centrifugation at 3,800 × g for 30 minutes at 4 °C. The pH of the supernatants was adjusted to 4.0 by adding hydrochloric acid prior to two extractions with an equal volume of ethyl acetate. The ethyl acetate fractions were collected and the solvent was removed by rotary evaporation to dryness at 40 °C. The residue was dissolved in 1 mL of methanol. The crude extract was subjected to a 0.45 μm Minisart filter unit and the collected filtrate was concentrated to 0.5 mL. Three microliters of the extract was injected into a C18 reverse-phase HPLC column (4.6 × 150 mm, Agilent Technologies), eluted with water in methanol (23:77, v/v, respectively; 0.1% formic acid) at a flow rate of 1 mL/minute in an Agilent Technologies 1260 Infinity system with a DAD G1315D VL detector.