DFPM root growth assay and subcellular localization of EDS1 isoforms in the root tissue

As described before [15], seeds were sterilized and germinated on half MS medium (half-strength Murashige and Skoog Basal Medium with Gamborg′s vitamins (M0404 Sigma), 0.05% MES, pH 5.7, 1% sucrose) plates with 0.8% phyto agar. After 2 days of stratification, seedlings were vertically grown for 10 days in 16 h/8 h long day conditions. On day 10, plants were transferred to new plates containing 10 μM DFPM. Subsequently, the root tip position was marked and root growth arrest was monitored after 6 d. All root growth experiments were repeated at least three times in independent experiments and representative images are shown.

For subcellular localization studies of EDS1-YFP isoforms within root cells, 5 day-old plant seedlings were exposed to 10 μM DFPM for 24 h and monitored using a Zeiss LSM 710 confocal microscope (Ex 514 nm/ Em 519–621 nm). Constant gain and pinhole values were used to observe pEDS1-EDS1-YFP, pEDS1-EDS1-YFP-NES and pEDS1-EDS1-YFP-NLS high expressor #A5. For pEDS1-EDS1-YFP-nes and the pEDS1-EDS1-YFP-NLS low expressor line #B2, an increased gain value was used to enhance the YFP signal.