Fig. 5: Effector-dependent NRG1–EDS1–SAG101 association requires a functional EDS1 EP domain. | Nature Communications

Fig. 5: Effector-dependent NRG1–EDS1–SAG101 association requires a functional EDS1 EP domain.

From: Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity

Fig. 5

a The Arabidopsis EDS1 (green)—SAG101 (purple) heterodimer crystal structure (PDB: 4NFU) with highlighted EP domain cavity essential for TNL-triggered cell death. An α-helix of the SAG101 EP domain (blue) and residues F419 (magenta) and H476 (orange) in EDS1 EP domain are shown as ribbon and spheres, respectively. b, c Roq1/XopQ-dependent cell death (b) and bacterial Xcv resistance (c) in leaves of Nb epss transiently expressing of C-terminally FLAG-tagged EDS1 wildtype and mutant variants with SAG101-FLAG and NRG1.1-SH or ADR1-L2-SH. Cell death was triggered by infiltrating Pf0-1 XopQ at 48 h after Agrobacteria infiltration; photos of leaves were taken at 24 h. F419E and H476Y mutations in the Arabidopsis EDS1 EP domain abrogated cell death and resistance. Experiments were performed three times independently, each with four replicates (leaf discs) (Tukey’s HSD, α = 0.001, n = 12). Error bars represent standard error of mean. Description of boxplots: minima—first quartile, maxima—third quartile, centre—median, whiskers extend to the minimum and maximum values but not further than 1.5 inter-quartile range from respective minima or maxima of the boxplot. Datapoints with the same colour come from one independent experiment. d IP followed by Western blot analysis to test the dependency of associations between Arabidopsis EDS1, SAG101 and NRG1.1 in Nb epss on a functional EDS1 EP domain cavity. Leaves of Nb epss were infiltrated with Agrobacteria to express FLAG-tagged EDS1 or its variants EDS1F419E and EDS1H476Y, SAG101-FLAG and NRG1.1-SH or PAD4-FLAG, with ADR1-L2-SH and GUS-FLAG as negative controls. At 2 dpi, Pf0-1 XopQ (OD600 = 0.3) was infiltrated, and the triggered samples were collected at 10 hpi. Following IP with α-HA agarose beads, input and IP fractions were probed with α-HA and α-FLAG antibodies. Roq1/XopQ-dependent association of NRG1.1-SH with EDS1-FLAG and SAG101-FLAG was abolished in samples with mutated EDS1 EP domain variants. Similar results were obtained in three independent experiments. Xcv Xanthomonas campestris pv. vesicatoria strain 85-10, XopQ (Pf0-1) effector tester strain of Pseudomonas fluorescens 0–1 delivering XopQ, WB Western blotting, IP immunoprecipitation, Nb epss Nicotiana benthamiana eds1a pad4 sag101a sag101b.

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