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The EDS1–PAD4–ADR1 node mediates Arabidopsis pattern-triggered immunity

Abstract

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1–PAD4–ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

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Fig. 1: RLCK-VII-7 subfamily members are required for LRR-RP-mediated immunity.
Fig. 2: PAD4, EDS1 and ADR1 helper NLRs are positive regulators of LRR-RP signalling.
Fig. 3: SOBIR1 associates with EDS1, PAD4 and ADR1 hNLRs to form a potential signalling node.

Data availability

All data are available within this article and its Supplementary Information. Proteomics data are available via the ProteomeXchange Consortium with the identifier PXD026120. MS data were searched against a combined database containing protein sequences from A. thaliana TAIR10_pep_20101214 (https://www.arabidopsis.org/download/index-auto.jsp?dir=%2Fdownload_files%2FProteins%2FTAIR10_protein_lists). Genomics data from A. thaliana accessions were obtained from the 1001 Genomes project (https://1001genomes.org/data-center.html) and mapped to the TAIR10 assembly of the genome (https://arabidopsis.org). Original gel blots are shown in Supplementary Fig. 1. Statistical analyses for all quantitative data are provided in Supplementary Tables 1, 2 and 5Source data are provided with this paper.

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Acknowledgements

This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants Nu 70/15-1 and ERA-CAPS-Grant SICOPID Nu 70/16-1 to T.N.; grant CRC-1101 to K.H., C.O., D.W., F.E.K. and T.N., and grant CRC-1403-414786233 to J.E.P. S. C. Saile was supported by the Reinhard Frank Stiftung (Project ‘helperless plant’). F.L., J.E.P., H.N., D.K. and D.W. were supported by the Max Planck Society. We thank S. Harter, M. Fechter, B. Löffelhardt and V. Scholz for assistance with cloning and genotyping; B. Kemmerling for statistical data evaluation; E. Chae for annotation information of NLRs; and P. Schulze-Lefert for the Lysobacter strain Root690.

Author information

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Authors

Contributions

R.N.P., M.H.A.J.J., B.P.H.J.T., J.L.D., F.E.K., J.E.P. and T.N. conceived and conceptualized the study. R.N.P., W.-L.W., M.H., S.R. and A.A.G. generated materials used in this study. R.N.P., K.F. and W.-L.W. performed the ethylene assays. R.N.P. and W.-L.W. performed the ROS assays. R.N.P., F.L., L.Z. and S. C. Saile performed co-IPs and western blots. R.N.P. performed the pathoassays. R.N.P., F.L. and C.H. performed the MAPK assays. A.J. performed the callose assays. R.N.P. and F.L. performed the RT–qPCR assays. S. C. Saile and F.E.K. performed the split-YFP assays. M.H. and J.-M.Z. performed the split firefly luciferase assays and analysed the data. F.W., C.O. and K.H. performed the FRET-FLIM assays and confocal microscopy and analysed the data. D.K. and D.W. performed the genetic analysis. Co-IP–MS experiments were designed by F.L., H.N. and J.E.P., executed by F.L. and A.H., and analysed by F.L., S. C. Stolze, H.N. and J.E.P. Statistical analysis was performed by R.N.P., F.L., S. C. Stolze and F.W. T.N. wrote the original draft of the paper. R.N.P., F.L., A.A.G., M.H.A.J.J., B.P.H.J.T., J.L.D., D.W., J.E.P. and T.N. reviewed and edited the paper.

Corresponding authors

Correspondence to Jane E. Parker or Thorsten Nürnberger.

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The authors declare no competing interests.

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Peer review information Nature thanks Gitta Coaker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 LRR-RP-mediated ethylene responses are dependent on RLCK-VII-7 kinases PBL30 and PBL31.

a, RLCK-VII mutant screen for positive regulators of LRR-RP signalling. n ≥ 6, each from 3 leaf pieces. Exact n values are shown in the graph. Data are from 2 independent experiments. Two-sided Wilcoxon rank sum pairwise tests with continuity correction were used to analyse significant differences between elicitor-treated Col-0 and the indicated mutant (*P ≤ 0.05, **P ≤ 0.01). b, Elicitor-induced ethylene production in Col-0 and RLCK-VII-7 mutants. n = 14, each from 3 leaf pieces. Data are from 3 independent experiments. Statistical differences between Col-0 and the indicated mutants were analysed using a Kruskal–Wallis test with a post hoc two-sided Steel-Dwass test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001). Centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR (IQR: the interquartile range between the 25th and the 75th percentile). Exact P values for all experiments are provided in Supplementary Table 5.

Source data

Extended Data Fig. 2 PBL31 activity in RLP23 signalling requires its kinase activity.

a, Ethylene accumulation in pbl30 pbl31 pbl32 complemented with wild-type PBL31-HA (PBL31) or the kinase-dead variant PBL31K201A-HA (PBL31K201A). Bars indicate mean ethylene response ± s.e.m. For PBL31K201A, n = 6; for all others, n = 9. A two-sided Welch’s t-test was used to analyse significant differences between Col-0 and the indicated line for the given elicitor treatment (**P ≤ 0.01, ***P ≤ 0.0001). Exact P values are provided in Supplementary Table 5. The experiment was repeated 3 times with similar results. b, PBL31 has autokinase activity that is abolished in the PBL31K201A mutant. Recombinant PBL31 and PBL31K201A were subjected to SDS–PAGE followed by anti-His protein blot. PBL31K201A runs near the predicted position for the tagged protein (57.4 kDa). The wild-type version migrates more slowly, consistent with it being auto-phosphorylated. Phosphorylation of the wild-type PBL31 was confirmed by treatment with calf intestinal phosphatase, which increased the migration rate of PBL31 but not PBL31K201A. The experiment was repeated 2 times with similar results. c, Anti-HA protein blot with material from plants used in a. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 3 Immune responses of RLCK-VII-7 mutants treated with LRR-RP-recognized and LRR-RK-recognized elicitors.

a, b, Elicitor-induced ROS production is impaired in pbl30 pbl31 pbl32. a, Total elicitor-induced ROS production over 30 min in Col-0, pbl30 pbl31, and pbl30 pbl31 pbl32. n = 48 leaf pieces from 3 independent experiments. For all panels pbl30 pbl31 is in orange, pbl30 pbl31 pbl32 is in pink. For a, c, h, statistical differences between Col-0 and the indicated mutants were analysed using a Kruskal–Wallis test with a post hoc two-sided Steel-Dwass test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001). b, Mean ROS production over time. Solid line, mean; shaded band, s.e.m.; n = 16 leaf pieces. c, Nlp20-induced callose deposition in Col-0 is dependent on PBL30 and PBL31. n ≥ 12 images from at least 3 leaves; exact n values are indicated on the graph. d, Nlp20-induced expression of PAD3, CYP71A13, and FMO1 is impaired in pbl30 pbl31 pbl32 plants. Bars indicate mean expression relative to EF-1α 6 h after mock or elicitor treatment determined by qRT–PCR. n = 8 biological replicates from 3 independent experiments. A two-sided Wilcoxon rank sum test with continuity correction was used to analyse significant differences between Col-0 and pbl30 pbl31 pbl32 for the given elicitor treatment (*P ≤ 0.05, **P ≤ 0.01). e, Relative fresh weight of 12 d-old Col-0 and pbl30 pbl31 pbl32 seedlings grown in the presence of flg22 or elf18. No significant differences were observed between Col-0 and pbl30 pbl31 pbl32 growth for any treatment (P > 0.05, two-sided Wilcoxon rank sum test with continuity correction). n = 8 biological replicates comprising 2 seedlings; for pbl30 pbl31 pbl32 treated with elf18, n = 7. f, MAP kinase activation in Col-0 and pbl30 pbl31 pbl32 treated with nlp20 or flg22 was analysed by immunoblot assay. Ponceau S-stained RUBISCO large subunit serves as a loading control. For gel source data, see Supplementary Fig. 1. g, Elicitor-induced defence against infection is impaired in pbl30 pbl31 and pbl30 pbl31 pbl32. Leaves were infiltrated with the indicated elicitor and challenged with Pst DC3000 infection after 24 h. Bacterial colonization was assessed at Day 0 and Day 3. n = 6 (Day 0) or 12 (Day 3) biological replicates comprising 2 leaf discs. Bars with different letters indicate significant differences of P ≤ 0.05 (Kruskal–Wallis test with post hoc two-sided Steel-Dwass test). No statistical differences were observed for Day 0. CFU, colony forming units. h, RLCK-VII-7 kinases are not required for an ETI response to Pst DC3000 AvrRps4 or Pst DC3000 AvrRpt2. n = 8 (Day 0) or 12 (Day 3) biological replicates comprising 2 discs; for pbl30 pbl31 pbl32 infected with Pst DC3000 AvrRpt2 (Day 3), n = 10. Growth on eds1 plants served as control. For box plots in a, c, centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR. For d, e, g, h bars indicate mean ± s.e.m. Experiments in b, c, eh were repeated at least three times with similar results. Exact P values for all quantitative experiments are provided in Supplementary Table 5.

Source data

Extended Data Fig. 4 PTI responses are partially dependent on PAD4 and EDS1.

a, Elicitor-induced ethylene production in pad4, eds1, sag101, EDS1 and EDS1LLIF lines. n = 14, each from 3 leaf pieces. Data are from 3 independent experiments. Statistical differences between Col-0 and the indicated mutants were analysed using a Kruskal–Wallis test with a post hoc two-sided Steel-Dwass test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001). Centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR. b, Nlp20-induced ethylene production is not dependent on EDS1 and PAD4 putative lipase activity. The eds1 pad4 is complemented with wild-type proteins (EDS1 PAD4) or variants harbouring mutations in their putative α/β-hydrolase catalytic residues (EDS1SDH PAD4S)19. n = 4, each from 3 leaf pieces. Statistical differences between Col-0 and the indicated mutants were analysed by two-sided Welch’s pairwise tests (**P ≤ 0.01). For be, bars indicate mean ± s.e.m. c, Thaxtomin A pretreatment enhances nlp20-induced ethylene responses in Col-0 but not in pad4 or pbl30 pbl31 pbl32 mutants. n = 4, each from 3 leaf pieces. Statistical differences between water- and thaxtomin A-treated samples were analysed using a Kruskal–Wallis test with a post hoc two-sided Steel-Dwass test (*P ≤ 0.05). d, Expression of PAD3, CYP71A13 and FMO1 6 h after elicitor or mock treatment, determined by qRT–PCR. n = 8 biological replicates from 3 independent experiments. A two-sided Wilcoxon rank sum test with continuity correction was used to analyse significant differences between Col-0 and pad4 for the given elicitor treatment (*P ≤ 0.05, **P ≤ 0.01). e, Relative fresh weight of 12 d-old Col-0 and pad4 seedlings grown in the presence of flg22 or elf18. No significant differences were observed between Col-0 and pad4 growth for any treatment (two-sided Wilcoxon rank sum test with continuity correction). n = 8 biological replicates comprising 2 seedlings; for Col-0 treated with elf18, n = 7. f, MAP kinase activation in Col-0 and eds1 pad4 sag101 treated with nlp20 or flg22 was analysed by immunoblot assay. Ponceau S-stained RUBISCO large subunit serves as a loading control. For gel source data, see Supplementary Fig. 1. Experiments in bf were performed at least three times with similar results. Exact P values for all quantitative experiments are provided in Supplementary Table 5.

Source data

Extended Data Fig. 5 Transcript and protein levels of immune-related genes in Col-0 and pad4.

a, Background levels of a set of immune-related genes in naive Col-0 and pad4. Relative expression was determined by qRT–PCR. Expression was normalized to EF-1α transcript and set relative to Col-0. n = 8 biological replicates from 3 independent experiments. Centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR. No significant differences were identified between Col-0 and pad4 (P > 0.05, two-sided Wilcoxon rank sum test with continuity correction). Exact P values are provided in Supplementary Table 5. b, Protein levels of FLS2, BAK1, MPK3, MPK4, and MPK6 are similar in Col-0 and pad4 plants. Leaves were taken from three 6-week-old plants (labelled 1-3) and endogenous protein levels were evaluated by protein blot. Ponceau S-stained RUBISCO large subunit serves as a loading control. For gel source data, see Supplementary Fig. 1. The experiment was repeated at least three times with similar results.

Source data

Extended Data Fig. 6 ADR1 helper NLRs are positive regulators of LRR-RP signalling.

a, Elicitor-induced ethylene production in helper NLR mutants. n = 13, each from 3 leaf pieces. Data are from 3 independent experiments. Col-0 is grey, adr1 triple is pink, nrg1 double is orange, and helperless is blue for all panels. For ac, f, statistical differences between Col-0 and the indicated mutant for the given elicitor treatment were analysed using a Kruskal–Wallis test with post hoc two-sided Steel-Dwass test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001). b, Total elicitor-induced ROS production over 30 min. n = 48 leaf discs from 3 independent experiments. c, Nlp20-induced callose deposition is reduced in adr1 triple and helperless mutants. n ≥ 12 images from at least 3 leaves. For Col-0 nlp20, n = 14 images; for adr1 triple mock, n = 15; for all others n = 16. d, Representative immunoblot for MAP kinase activation in Col-0 and helperless treated with nlp20 or flg22. Ponceau S-stained RUBISCO large subunit serves as a loading control. For gel source data, see Supplementary Fig. 1. e, Relative fresh weight of 12 d-old Col-0 and helperless seedlings grown in the presence of flg22 or elf18 (n = 8 biological replicates comprising 2 seedlings). No statistical differences between Col-0 and helperless were identified for each elicitor treatment (P > 0.05, two-sided student’s t-test). f, Elicitor-induced defence against infection is impaired in adr1 triple and helperless mutants. Leaves were infiltrated with the indicated elicitor and challenged with Pst DC3000 24 h after infiltration. Bacterial colonization was assessed at Day 0 and Day 3. n = 6 (Day 0) or n = 12 (Day 3) biological replicates comprising 2 leaf discs. Bars with different letters indicate significant differences of P ≤ 0.05 (Kruskal–Wallis test with post hoc two-sided Steel-Dwass test). No statistical differences were observed for Day 0. CFU, colony forming units. For box plots in ac, centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR. For e, f, bars indicate mean ± s.e.m. Experiments in cf were performed at least three times with similar results. Exact P values for all quantitative experiments are provided in Supplementary Table 5.

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Extended Data Fig. 7 Co-immunoprecipitation, split-YFP and split-luciferase complementation assays suggest that SOBIR1 is associated with multiple downstream signalling components.

a, PBL31, ADR1, EDS1, and PAD4 associate with SOBIR1 in a nlp20-independent manner. The indicated proteins were transiently expressed in Nicotiana benthamiana. Leaves treated with nlp20 or water (mock) for 10 min were subjected to co-immunoprecipitation with GFP-trap beads. The proteins were not co-immunoprecipitated with a GFP-fused membrane protein (LTI6b56). The experiment was performed twice with similar results. b, Pull-down of GFP and SOBIR1-GFP transiently co-expressed with ADR1-HA, ADR1-L1-HA or ADR1-L2-HA. Plants transiently expressing the different proteins were subjected to co-immunoprecipitation using GFP-trap beads and subsequently analysed by protein blot using tag-specific antisera. ADR1-L1-HA and ADR1-L2-HA were co-immunoprecipitated at least three times with similar results; ADR1-HA was tested once. c, BiFC between SOBIR1 and the ADR1 isoforms confirms constitutive interaction of SOBIR1 with ADR1-L1 and ADR1-L2 at the plasma membrane. Scale bar indicates 20 μm. The experiment was performed at least three times with similar results. d, Protein levels of the transiently expressed proteins in BiFC experiments shown in panel c. e, Split luciferase complementation assays confirm the interaction of SOBIR1 and ADR1-L1. Bars indicate mean relative luciferase activity ± s.e.m.: n = 8 leaf discs from 4 leaves. The experiment was performed three times with similar results. f, Protein levels of the transiently expressed proteins in split luciferase experiments shown in panel e. Co-expression of the SOBIR1 and PBL31 constructs led to cell death and low protein abundance. The experiment was performed twice with similar results. For gel source data, see Supplementary Fig. 1.

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Extended Data Fig. 8 FRET-FLIM analysis demonstrates association of PBL31-GFP with SOBIR1, ADR1-L1 and EDS1.

a, b, Representative confocal images show co-localization of (a) SOBIR1-GFP or (b) PBL31-GFP with RFP fusions of ADR1, ADR1-L1, ADR1-L2, EDS1, PAD4, SAG101, (a) PBL31, or (b) SOBIR1 at the PM in transiently transformed N. benthamiana leaf cells. Plots show the GFP and RFP fluorescence intensity distribution across the PM in the indicated regions (white bars). Scale bars indicate 10 μm. This experiment was repeated three times with similar results. c, FRET-FLIM reveals spatial proximity of PBL31-GFP with ADR1-L1-RFP, EDS1-RFP, EDS1-RFP + PAD4-HA, PAD4-RFP + EDS1-HA and SOBIR1-RFP. Membrane-associated protein NPH3S744A serves as control. n ≥ 11 measurements from at least 3 biological replicates. Exact n values are shown below the boxes. Statistical differences in fluorescent lifetime from PBL31-GFP were analysed using a Kruskal–Wallis test with post hoc two-sided Steel-Dwass test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001). Exact P values are provided in Supplementary Table 5. Centre line: median, bounds of box: 25th and 75th percentiles, whiskers: 1.5 * IQR.

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Extended Data Fig. 9 SOBIR1 specifically co-purifies with YFP-PAD4 in Arabidopsis leaves in mock and nlp20-triggered conditions.

a, b, Arabidopsis pad4-1/sag101-3 plants complemented with pPAD4::YFP-gPAD4 (YFP-PAD4) or pSAG101::gSAG101-YFP (SAG101-YFP) or 35S::YFP (YFP) in wild-type Col-0 background were used for immunoprecipitation (IP) assays. Volcano plots show normalized abundances (LFQ, log2 scale) of proteins detected in mass-spectrometry (MS) analyses after IP of total protein extracts from 4.5-week-old Arabidopsis pad4-1/sag101-3 complementation lines or YFP control line infiltrated with a, nlp20 for 10 min, (b, upper panels) DMSO (Mock) for 3 h, and (b, lower panels) nlp20 for 3 h. Red dots indicate proteins enriched in YFP-PAD4 vs YFP samples (log2(YFP-PAD4 vs YFP) ≥ 1, left) and YFP-PAD4 vs SAG101-YFP samples (log2(YFP-PAD4 vs SAG101-YFP) ≥ 1, right), using permutation-based FDR = 0.05. Graphs represent significantly enriched peptides from four independent experiments (n = 4 per genotype per treatment). As shown for 10 min treatments (Fig. 3b), specific enrichment of the two functional Col-0 EDS1 isoforms (EDS1a and EDS1b) was detected in both YFP-PAD4 and SAG101-YFP samples, with EDS1b preferentially enriched following YFP-PAD4 pull-down. c, Representative immunoblot analyses to check test protein quality for IP quality in lines used for MS/MS analyses. Total protein extracts (IP inputs) from YFP-PAD4, SAG101-YFP and YFP lines infiltrated with DMSO (mock) or nlp20 for 10 min. Inputs were subsequently immunoprecipitated using GFP-trap agarose beads and analysed by mass spectrometry. The analyses were repeated four times for both 10 min and 3 h treatments with similar results (n = 8 per genotype per treatment). d, e, Nlp20 treatment triggered immune responses at early (10 min) and late (3 h) time points in samples used for IP MS/MS analyses. d, Total protein extracts from Col‐0, YFP-PAD4, SAG101-YFP and YFP lines infiltrated with DMSO (mock) and nlp20 for 10 min were analysed on immunoblots using an anti‐p44/42‐ERK antibody. The identity of individual phosphorylated (p)-MAPKs, as determined by their mobility, is indicated by arrows. The analysis was repeated four times with similar results (n = 4 samples per genotype per treatment). For gel source data, see Supplementary Fig. 1. e, PAD3 transcript levels were determined by qRT–PCR at 3 h after mock (DMSO) or nlp20 treatment of the indicated genotypes. Relative expression was normalized to UBQ5 and set to Col-0 mock samples. Bars indicate mean expression ± s.e.m. (n = 12 biological replicates from 4 independent experiments). Statistical differences between Col-0 and the indicated genotypes were analysed using Kruskal–Wallis with post hoc two-sided pairwise comparisons using Wilcoxon rank sum test with a Benjamini-Hochberg correction (*P ≤ 0.05, **P ≤ 0.01). Exact P values are provided in Supplementary Table 5.

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Extended Data Fig. 10 Classification of LRR-RPs, LRR-RKs and NLRs according to genetic conservation in Arabidopsis accessions.

a, Reads from 80 Arabidopsis accessions were mapped to the reference genome of Col-0. Genes were categorised as being conserved, having complex patterns of variation or exhibiting presence/absence polymorphisms according to the distribution of large-scale polymorphisms across all accessions as inferred from stringent read mappings. Criteria for categorization are detailed in the Methods. The numbers of genes falling into each category are provided in the corresponding bars. b, LRR-RP genes classified as in a. Genes encoding known immune receptors are indicated in bold.

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Supplementary information

Supplementary Figure 1

This file contains all uncropped blots and gel images.

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Supplementary Table 1

MS/MS and statistical analysis for GFP, YFP-PAD4, and SAG101-YFP treated for 10 min.

Supplementary Table 2

MS/MS and statistical analysis for GFP, YFP-PAD4, and SAG101-YFP treated for 3 h.

Supplementary Table 3

Arabidopsis lines used in this study.

Supplementary Table 4

Quantitative reverse transcription-PCR (qRT-PCR) primers used in this study.

Supplementary Table 5

Statistical summary.

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Pruitt, R.N., Locci, F., Wanke, F. et al. The EDS1–PAD4–ADR1 node mediates Arabidopsis pattern-triggered immunity. Nature (2021). https://doi.org/10.1038/s41586-021-03829-0

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