Fig. 3: Early effector-dependent NRG1 association with EDS1 and SAG101 in Arabidopsis TNLRRS1–RPS4 triggered ETI. | Nature Communications

Fig. 3: Early effector-dependent NRG1 association with EDS1 and SAG101 in Arabidopsis TNLRRS1–RPS4 triggered ETI.

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

Fig. 3

a Dotplot of normalised abundance values (label-free quantification (LFQ), log2-scaled) for proteins detected in liquid chromatography–mass spectrometry (LC–MS) analyses after α-GFP immunoprecipitation (IP) of PAD4-YFP or SAG101-YFP from total leaf extracts of respective complementation lines pPAD4:YFP-PAD4 and pSAG101:SAG101-YFP (both Col pad4 sag101 background) after infiltration with effector tester stra5in of Pseudomonas fluorescens 0-1 (Pf0-1) avrRps4 (6 hpi, OD600 = 0.2). ADR1s are specifically enriched in PAD4-YFP IP samples, whereas NRG1s are more abundant in the SAG101-YFP IP samples. Samples were collected from four independent experiments. b Dotplot of LFQ values for proteins detected in LC–MS analyses after IP of EDS1-YFP and TRB1-GFP from nuclei-enriched extracts of corresponding Arabidopsis complementation lines infiltrated with Pseudomonas syringae pv. tomato DC3000 (Pst) avrRps4 (8 hpi, OD600 = 0.1). NRG1.1 and NRG1.2 are specifically enriched in EDS1-YFP samples. Samples were collected from four independent experiments. c α-GFP probed immunoblots of nuclei-enriched extracts from leaves of the Arabidopsis pEDS1:EDS1-YFP complementation line (Col eds1-2 background) infiltrated with Pst avrRps4 (OD600 = 0.1, 8 hpi). Extracts were resolved using native gel filtration. Arrows below protein markers indicate position of the corresponding peak. Numbers refer to column fractions. EDS1 forms stable ~100–600 kDa complexes. The experiment was conducted three times with similar results. d LC–MS analysis of eluates after α-FLAG IP of total leaf extracts from Arabidopsis Ws-2 n2 pNRG1.2:NRG1.2-HF complementation line infiltrated with Pf0-1 empty vector (EV) or Pf0-1 avrRps4 (4 hpi, OD600 = 0.3). Peptides corresponding to EDS1 and SAG101 were observed in eluates only after Pf0-1-mediated delivery of avrRps4. This result was observed in two independent experiments. e Western blot (WB) analysis of eluates from (d). Asterisk indicates a nonspecific band on the α-EDS1 blot for input samples. The analysis was performed on total leaf extracts and was conducted four times with similar results. Association of EDS1 with NRG1.2-HF was observed only after Pf0-1-mediated delivery of avrRps4. Ponceau S staining shows similar protein loading in input samples on the blot.

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