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. 2021 Jun 7;12:3335. doi: 10.1038/s41467-021-23614-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Sample collection scheme for experiments in (b) and (c). Roq1-dependent cell death is restored in Nicotiana benthamiana (Nb) eds1 pad4 sag101a sag101b (Nb epss) signalling deficient mutant by expressing Arabidopsis EDS1, SAG101 and NRG1.1 via Agrobacteria infiltration 48 h before XopQ effector delivery. Pf0-1 XopQ is syringe-infiltrated (OD₆₀₀ = 0.3) to deliver the effector and to study TNL signalling events in a time-resolved manner up to 24 hpi. b Macroscopic symptoms and quantification of XopQ-triggered cell death at 24 hpi after infiltrating Pf0-1 XopQ in leaves of Nb epss expressing Arabidopsis EDS1-FLAG, SAG101-FLAG, NRG1.1-3xHA-StrepII (NRG1.1-SH) or ADR1-L2-3xHA-StrepII (ADR1-L2-SH). Pf0-1 with empty vector (Pf0-1) served as a “no-ETI” control. Cell death was quantified in electrolyte leakage assays 6 h after harvesting leaf discs (24 hpi with Pf0-1 XopQ). The experiment was performed three times independently, each with four replicates (leaf discs) (Tukey’s HSD, α = 0.001, n = 12). Transiently expressed Arabidopsis EDS1, SAG101 and NRG1.1 proteins are functional in Pf0-1 XopQ triggered (Roq1) cell death. 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 interquartile range from the respective minima or maxima of the boxplot. Datapoints with the same colour come from one independent experiment. c Coimmunoprecipitation assay followed by Western blotting to test for XopQ-triggered associations between Arabidopsis NRG1.1-SH and FLAG-tagged EDS1 or SAG101 in Nb epss according to the infiltration scheme in (a). NRG1.1-SH or ADR1-SH were enriched using α-HA agarose beads, and presence of FLAG-tagged EDS1, SAG101 or GUS was tested by probing blots with α-FLAG antibodies. IP assays were conducted three times independently with similar results. NRG1.1 association with EDS1 and SAG101 requires Roq1/XopQ ETI activation. Pf0-1 effector tester strain of Pseudomonas fluorescens 0–1 with empty vector, Pf0-1 XopQ (in a) or 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.