Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana

Abstract

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor–like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Elevations in [Ca2+]cyt in A. thaliana seedlings treated with Pseudomonas LPS.
Figure 2: LPS-triggered PTI responses in A. thaliana.
Figure 3: Infection of A. thaliana with P. syringae.
Figure 4: A. thaliana PTI responses to LPS from various bacteria.
Figure 5: Transient expression of LORE in tobacco.
Figure 6: A. thaliana PTI responses to various part structures of Pseudomonas LPS.

References

  1. 1

    Jones, J.D. & Dangl, J.L. The plant immune system. Nature 444, 323–329 (2006).

    CAS  PubMed  Google Scholar 

  2. 2

    Boller, T. & Felix, G. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379–406 (2009).

    CAS  PubMed  Google Scholar 

  3. 3

    Shiu, S. & Bleecker, A. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. USA 98, 10763–10768 (2001).

    CAS  PubMed  Google Scholar 

  4. 4

    Shiu, S. & Bleecker, A. Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol. 132, 530–543 (2003).

    CAS  PubMed  Google Scholar 

  5. 5

    Zipfel, C. Plant pattern-recognition receptors. Trends Immunol. 35, 345–351 (2014).

    CAS  PubMed  Google Scholar 

  6. 6

    Wan, J. et al. LYK4, a lysin motif receptor-like kinase, is important for chitin signaling and plant innate immunity in Arabidopsis. Plant Physiol. 160, 396–406 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Cao, Y. et al. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife doi:10.7554/eLife.03766 (2014).10.7554/eLife.03766

  8. 8

    Knirel, Y.A., Bystrova, O.V., Kocharova, N.A., Zähringer, U. & Pier, G.B. Conserved and variable structural features in the lipopolysaccharide of Pseudomonas aeruginosa. J. Endotoxin Res. 12, 324–336 (2006).

    CAS  PubMed  Google Scholar 

  9. 9

    Zipfel, C. & Felix, G. Plants and animals: a different taste for microbes? Curr. Opin. Plant Biol. 8, 353–360 (2005).

    CAS  PubMed  Google Scholar 

  10. 10

    Schroeder, T.H. et al. CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-κB translocation. Proc. Natl. Acad. Sci. USA 99, 6907–6912 (2002).

    CAS  PubMed  Google Scholar 

  11. 11

    McIsaac, S.M., Stadnyk, A.W. & Lin, T.-J. Toll-like receptors in the host defense against Pseudomonas aeruginosa respiratory infection and cystic fibrosis. J. Leukoc. Biol. doi:10.1189/jlb.0811410 (2012).10.1189/jlb.0811410

  12. 12

    Tan, Y. & Kagan, J.C. A cross-disciplinary perspective on the innate immune responses to bacterial lipopolysaccharide. Mol. Cell 54, 212–223 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Shi, J. et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514, 187–192 (2014).

    CAS  PubMed  Google Scholar 

  14. 14

    Newman, M.A., Sundelin, T., Nielsen, J.T. & Erbs, G. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front. Plant Sci. 4, 139 (2013).

    PubMed  PubMed Central  Google Scholar 

  15. 15

    Silipo, A. et al. Glyco-conjugates as elicitors or suppressors of plant innate immunity. Glycobiology 20, 406–419 (2010).

    CAS  PubMed  Google Scholar 

  16. 16

    Zeidler, D. et al. Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. Proc. Natl. Acad. Sci. USA 101, 15811–15816 (2004).

    CAS  PubMed  Google Scholar 

  17. 17

    Silipo, A. et al. The elicitation of plant innate immunity by lipooligosaccharide of Xanthomonas campestris. J. Biol. Chem. 280, 33660–33668 (2005).

    CAS  PubMed  Google Scholar 

  18. 18

    Bedini, E. et al. Structure-dependent modulation of a pathogen response in plants by synthetic O-antigen polysaccharides. J. Am. Chem. Soc. 127, 2414–2416 (2005).

    CAS  PubMed  Google Scholar 

  19. 19

    Madala, N.E., Molinaro, A. & Dubery, I.A. Distinct carbohydrate and lipid-based molecular patterns within lipopolysaccharides from Burkholderia cepacia contribute to defense-associated differential gene expression in Arabidopsis thaliana. Innate Immun. 18, 140–154 (2012).

    CAS  PubMed  Google Scholar 

  20. 20

    Ranf, S., Eschen-Lippold, L., Pecher, P., Lee, J. & Scheel, D. Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns. Plant J. 68, 100–113 (2011).

    CAS  PubMed  Google Scholar 

  21. 21

    Bitter, W. et al. Species-specific functioning of the Pseudomonas XcpQ secretin: role for the C-terminal homology domain and lipopolysaccharide. J. Bacteriol. 189, 2967–2975 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Kocíncová, D. & Lam, J.S. Structural diversity of the core oligosaccharide domain of Pseudomonas aeruginosa lipopolysaccharide. Biochemistry (Mosc.) 76, 755–760 (2011).

    Google Scholar 

  23. 23

    Sánchez Carballo, P.M., Rietschel, E.T., Kosma, P. & Zähringer, U. Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4. Eur. J. Biochem. 261, 500–508 (1999).

    PubMed  Google Scholar 

  24. 24

    Ranf, S. et al. Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana. Mol. Plant 5, 115–130 (2012).

    CAS  PubMed  Google Scholar 

  25. 25

    Vaid, N., Pandey, P.K. & Tuteja, N. Genome-wide analysis of lectin receptor-like kinase family from Arabidopsis and rice. Plant Mol. Biol. 80, 365–388 (2012).

    CAS  PubMed  Google Scholar 

  26. 26

    Zipfel, C. et al. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428, 764–767 (2004).

    CAS  PubMed  Google Scholar 

  27. 27

    Xing, S., Li, M. & Liu, P. Evolution of S-domain receptor-like kinases in land plants and origination of S-locus receptor kinases in Brassicaceae. BMC Evol. Biol. 13, 69 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Samuel, M.A. et al. Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis. Plant Physiol. 147, 2084–2095 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Mamat, U. et al. WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis. J. Biol. Chem. 284, 22248–22262 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Alexander, C. & Rietschel, E.T. Bacterial lipopolysaccharides and innate immunity. J. Endotoxin Res. 7, 167–202 (2001).

    CAS  PubMed  Google Scholar 

  31. 31

    Miller, S.I., Ernst, R.K. & Bader, M.W. LPS, TLR4 and infectious disease diversity. Nat. Rev. Microbiol. 3, 36–46 (2005).

    CAS  PubMed  Google Scholar 

  32. 32

    Zähringer, U. et al. NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus. J. Biol. Chem. 289, 23963–23976 (2014).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Ittig, S. et al. The lipopolysaccharide from Capnocytophaga canimorsus reveals an unexpected role of the core-oligosaccharide in MD-2 binding. PLoS Pathog. 8, e1002667 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Silipo, A. et al. The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis. ChemBioChem 9, 896–904 (2008).

    CAS  PubMed  Google Scholar 

  35. 35

    Ciesielski, F., Griffin, D.C., Rittig, M., Moriyón, I. & Bonev, B.B. Interactions of lipopolysaccharide with lipid membranes, raft models - a solid state NMR study. Biochim. Biophys. Acta 1828, 1731–1742 (2013).

    CAS  PubMed  Google Scholar 

  36. 36

    Ciesielski, F., Davis, B., Rittig, M., Bonev, B.B. & O'Shea, P. Receptor-independent interaction of bacterial lipopolysaccharide with lipid and lymphocyte membranes; the role of cholesterol. PLoS ONE 7, e38677 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Park, B.S. et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458, 1191–1195 (2009).

    CAS  PubMed  Google Scholar 

  38. 38

    Nürnberger, T., Brunner, F., Kemmerling, B. & Piater, L. Innate immunity in plants and animals: striking similarities and obvious differences. Immunol. Rev. 198, 249–266 (2004).

    PubMed  Google Scholar 

  39. 39

    Takasaki, T. et al. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403, 913–916 (2000).

    CAS  PubMed  Google Scholar 

  40. 40

    Ivanov, R., Fobis-Loisy, I. & Gaude, T. When no means no: guide to Brassicaceae self-incompatibility. Trends Plant Sci. 15, 387–394 (2010).

    CAS  PubMed  Google Scholar 

  41. 41

    Knight, M.R., Campbell, A.K., Smith, S.M. & Trewavas, A.J. Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352, 524–526 (1991).

    CAS  PubMed  Google Scholar 

  42. 42

    Gómez-Gómez, L., Felix, G. & Boller, T. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana. Plant J. 18, 277–284 (1999).

    PubMed  Google Scholar 

  43. 43

    Kunze, G. et al. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496–3507 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Westphal, O. & Jann, K. in Methods Carbohydr. Chem. vol. 5. (eds. Whistler. R. & Wolfan, M.) 83–91 (Academic Press, 1965).

  45. 45

    Galanos, C., Lüderitz, O. & Westphal, O. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245–249 (1969).

    CAS  PubMed  Google Scholar 

  46. 46

    Zähringer, U., Salvetzki, R., Wagner, F., Lindner, B. & Ulmer, A.J. Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes. J. Endotoxin Res. 7, 133–146 (2001).

    PubMed  Google Scholar 

  47. 47

    Salathia, N. et al. Indel arrays: an affordable alternative for genotyping. Plant J. 51, 727–737 (2007).

    CAS  PubMed  Google Scholar 

  48. 48

    Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3, e3647 (2008).

    PubMed  PubMed Central  Google Scholar 

  49. 49

    Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank W. Bitter (Utrecht University) for P. alcaligenes 537 bacterial mass; B.A. Dmitriev (Gamaleya Research Institute of Epidemiology and Microbiology) for LPS of P. aeruginosa Fisher type 1; E. Glawischnig (TU München) for E. salsugineum ecotype Shandong plants; K. Kawahara (The Kitasato Institute) for LPS of Burkholderia species; M. Knight (Durham University) for Col-0AEQ A. thaliana; U. Mamat (Research Center Borstel) for E. coli strain KPM 53; M. Quint (IPB Halle) for C. rubella seeds; K. Rudolph (Universität Göttingen) for P. syringe pv. apii bacterial mass; S.N. Senchenkova for the isolation of X. campestris LPS; G. Stacey (University of Missouri) for T-DNA–insertion lines cerk1-2 and lyk4; C. Hemetsberger for assistance with confocal imaging; N. Bauer, B. Buske, B. Kölbl, B. Kunz, H. Moll and U. Schombel for technical assistance; H. Schempp for discussions; and C. Schwechheimer for critical reading of the manuscript. Supported by the German Research Foundation (through Priority Program SPP1212 to J.L. and D.S.; and Collaborative Research Centre SFB924/TP-B10 to S.R.).

Author information

Affiliations

Authors

Contributions

S.R., M.S., T.I., L.W. and J.L. performed experiments; S.R., Y.A.K., P.M.S.-C. and U.Z. prepared LPS; S.R. and D.S. initiated the project; S.R. designed plant experiments; N.G. and U.Z. designed LPS purification and chemical dissection experiments; S.R., N.G. and J.L. analyzed the data; S.R., N.G., J.L. and R.H. interpreted data; S.R., N.G., R.H., U.Z., J.L. and D.S. wrote the manuscript; and all authors discussed the results and approved of the manuscript.

Corresponding author

Correspondence to Stefanie Ranf.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 LPS-induced elevations in [Ca2+]cyt in A. thaliana cerk1-2, lyk4, fls2-26, efr-1 and bak1-4 mutants and after treatment with proteinase K, calcium channel blocker or protein kinase inhibitor.

(a) [Ca2+]cyt in Col‑0AEQ control and indicated mutant seedlings treated with LPS from P. aeruginosa PAO1 or H4 (50 μg/ml), with reconstitution of aequorin with 5 µM coelenterazine‑h; results are presented as luminescence counts per second relative to total luminescence counts remaining (L/Lmax). Black bars represent maximum LPS-induced elevations in [Ca2+]cyt in the indicated mutant line, while grey bars represent maximum LPS-induced elevations in [Ca2+]cyt in the respective Col‑0AEQ control. (b) Col‑0AEQ seedlings were treated with LPS (50 µg/ml) or flg22 or elf18 peptides (1 µM). LPS preparations (30 µl of 2 mg/ml) and peptide solutions (30 µl of 40 µM) were incubated overnight at 60 °C with proteinase K (10 µg) or without as control and were heat-inactivated for 15 min at 95 °C. Maximum [Ca2+]cyt induced by control-treated elicitor samples are shown in comparison to maximum [Ca2+]cyt induced by proteinase K-treated elicitor samples at peak maximum (LPS) or at time of peak maximum in the control (flg22 and elf18), with reconstitution of aequorin with 5 µM coelenterazine‑h. (c) Col‑0AEQ seedlings were treated with 10 mM LaCl3, 10 µM K‑252a or DMSO as control for 30 min before application of LPS from P. aeruginosa PAO1 or H4 (50 μg/ml). Maximum LPS-induced elevations in [Ca2+]cyt in DMSO-treated controls are shown in comparison to LPS-induced elevations in [Ca2+]cyt at the same time in inhibitor-treated samples, with reconstitution of aequorin with 10 µM native coelenterazine. (d) Scheme of LPS structure showing a representative core region and lipid A of Pseudomonas aeruginosa. Black dashed lines indicate truncations in rough mutants, grey dotted lines non-stoichiometric modifications. Ara4N, 4-amino-4-deoxy-L-arabinose; Cm, carbamoyl; Etn, ethanolamine; GalN, galactosamine; Glc, glucose; GlcN, glucosamine; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LOS, lipooligosaccharide; OPS, O-polysaccharide; P, phosphate; Rha, L-rhamnose. Data are representative of two experiments with similar results (b) or one experiment (a,c) (mean and s.d. of n = 6, 6; 4, 6; 4, 6; 4, 9; 6, 4 (left panel, left to right) and n = 9, 9; 6, 6; 6, 6; 6, 9; 9, 5 (right panel, left to right) seedlings per genotype and treatment in a, or n = 4 seedlings per treatment in b,c).

Supplementary Figure 2 ROS production, MPK activation and callose deposition in response to LPS in A. thaliana.

(a) ROS accumulation in leaf discs of Col‑0 control and rbohD mutant plants treated with LPS from P. aeruginosa H4 (left panel) or P. syringae pv. tomato DC3000 (right panel) (10 µg/ml); results were calculated by subtraction of ROS of untreated control samples and are presented as relative light units (RLU). (b) Immunoblot analysis (IB) of phosphorylated MPK6, MPK3 and MPK4 in A. thaliana Col‑0, mpk3-1 and mpk6-3 seedlings at 5 or 10 min (above lanes) after treatment with LPS from P. aeruginosa H4 or P. syringae pv. tomato DC3000 (20 μg/ml), with chitin (50 μg/ml) or with flg22 (100 nM); below, amido black staining of total proteins (loading control). (c) Col‑0 seedlings were treated for 24 h with LPS from P. aeruginosa H4 or water (control). Callose deposition was visualized by fluorescence microscopy after aniline blue staining. Four individual cotyledons per treatment are shown (scale bar = 200 µm). Data are representative of two experiments (a,b; mean and s.e.m. of n = 8 leaf discs from four plants per genotype in a) or three experiments (c) with similar results.

Supplementary Figure 3 LPS-induced elevations in [Ca2+]cyt and activation of MAPKs in A. haliana lore, sd1-29 and sd1-23 mutants.

(a) [Ca2+]cyt over time in Col‑0AEQ control and different lore mutant seedlings treated with LPS from P. aeruginosa H4 (50 µg/ml), with reconstitution of aequorin with 20 µM native coelenterazine. Full figure of Figure 2a. (b) Schematic representation of SD129 and SD123 gene structure. Black boxes indicate exons and white boxes indicate untranslated regions (UTR). Arrowheads show location of lore point mutations and white arrows T‑DNA insertion sites. (c) LORE protein scheme showing predicted conserved domains and location of lore loss-of-function mutations. SP, signal peptide; TM, transmembrane; EGF, epidermal growth factor; PAN, plasminogen-apple-nematode; DUF, domain of unknown function. (d) Expression of SD129 and SD123 genes was assessed by quantitative real-time RT-PCR (before and after the T-DNA insertion site) in two Col‑0 control, sd1‑29 and sd1‑23 seedling pools each (labelled as cDNA (1) and (2)) or by semi-quantitative RT-PCR (across the T-DNA insertion site) using the primers (Supplementary Table 2) indicated by colored arrows. Expression of the EF1a gene served as constitutive control. (e) [Ca2+]cyt over time in Col‑0AEQ, sd129AEQ and lore‑1 seedlings treated with LPS from P. aeruginosa H4 (15 µg/ml), with reconstitution of aequorin with 5 µM coelenterazine‑h. (f) Immunoblot analysis (IB) of phosphorylated MPK6, MPK3 and MPK4 in Col‑0AEQ, lore‑1, Col‑0, sd1‑29 and sd1‑23 A. thaliana seedlings at various times (above lanes) after treatment with different LPS (20 μg/ml), chitin (50 μg/ml) or water (control); below, amido black staining of total proteins (loading control). Full version of Figure 2f. Data are representative of two experiments with similar results (a,d,e,f; mean and s.d. of n = 7, 7; 7, 3; 7, 7; 7, 9 (left to right) seedlings per genotype with Col‑0AEQ controls identical in all graphs in a, or n = 10, 7, 10 (top down) seedlings per genotype in e).

Supplementary Figure 4 Infection of A. thaliana with P. syringae.

(a) Bacterial growth in A. thaliana Col‑0AEQ control, lore‑1 mutant and complementation lines (CL) on days 0 and 3 after spray inoculation with Pst DC3000 (1 × 108 colony-forming units (CFU)/ml inoculum). Different letters indicate significant difference with P < 0.05, same letters indicate no significant difference (two-way analysis of variance (ANOVA) (genotype × treatment) and Bonferroni’s multiple-comparison post-test). (b,c) Bacterial growth in A. thaliana Col‑0AEQ control and lore‑1 mutant (b) or Col‑0 control and sd1‑29 mutant (c) treated with the indicated LPS samples (25 μg/ml) or water as control 1 d before infiltration of Pst DC3000 (1 × 105 CFU/ml inoculum), assessed on days 0 and 3. Different letters indicate significant difference with P < 0.05 at day 3, same letters indicate no significant difference (one-way ANOVA and Tukey’s multiple-comparison post-test). Data are representative of three experiments with similar results (a) or one experiment (b,c) (mean and s.d. of n = 12 (a); n = 6 (b); or n = 4 (sd1‑29, day 3) or n = 6 (all others) (c) leaf samples randomly obtained from four (a) or three (b,c) plants per genotype).

Supplementary Figure 5 Phylogenetic distribution of LORE and functional analysis of LPS sensing in selected plant species.

(a) Phylogram of SD1‑RLKs from A. thaliana and diverse other plant species. Protein BLAST was performed against diverse plant species using LORE amino acid sequence as query. Sequence data sources: TAIR10, Ensembl Plants, BOLbase, Sol Genomics Network and NCBI databases. Plant families (species): Brassicaceae (Arabidopsis thaliana, Arabidopsis lyrata, Brassica rapa, Brassica oleracea, Capsella rubella, Eutrema salsugineum), Fabaceae (Glycine max, Medicago truncatula), Solanaceae (Nicotiana benthamiana, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum), Poaceae (Hordeum vulgare, Oryza sativa, Zea mays), Salicaceae (Populus trichocarpa). Sequences of A. thaliana SD1-RLKs4, RLKs FLS2 and CERK1, and the best LORE BLAST hits (max. 3 from each species) were aligned using ClustalW. A phylogenetic tree was constructed using the Maximum Likelihood method implemented in MEGA6. Bootstrapping values (1000 replicates; >50%) are shown next to the branches. Branch lengths indicate the number of substitutions per site. LORE (marked with red filled dot) and its putative orthologs are highlighted in red. A subclade apparently specific to the Brassicaceae is highlighted in blue. The paralog most similar to LORE, SD1‑23, is marked with a blue diamond, Brassica SRKs (Self-incompatibility Receptor Kinases) with black triangles, and tobacco SD1-RLKs with grey squares. (b) ROS accumulation in leaf discs of E. salsugineum (left) and C. rubella (right) (marked with red open circles in a) treated with the indicated LPS samples (25 µg/ml); results were normalized to ROS before LPS treatment and were calculated by subtraction of ROS of untreated control samples and are presented as relative light units (RLU). (c) ROS accumulation in leaf discs of N. tabacum (left) and N. benthamiana (right) treated with the indicated LPS preparations (50 µg/ml) or flg22 (1 µM). (d) [Ca2+]cyt over time in aequorin-expressing N. tabacum seedlings treated with the indicated LPS preparations (50 µg/ml) or flg22 (1 µM) after 2 min measurement of the resting [Ca2+]cyt. (e) [Ca2+]cyt in aequorin-expressing N. tabacum seedlings 1 min before elicitation (grey bars) and maximum elevation (black bars) after application of the indicated LPS preparations (50 µg/ml), flg22 (1 µM) or water (control). (f) Epidermal N. benthamiana cell expressing LORE‑Km-mCherry before and ~15 min after treatment with 1 M NaCl for plasmolysis. Scale bars, 25 µm. Due to water loss in the hypertonic NaCl solution, the protoplast including the plasma membrane is retracted from the cell wall. Data are representative of three (b) or two experiments (c) with similar results or one experiment (d-f) (mean and s.e.m. of n = 8 leaf discs from four (b) or three (c-e) plants per genotype; or mean and s.d. of n = 3 (Pst LPS) or n = 4 (all other) seedlings per treatment). Data in (d) and (e) are from the same experiment with (d) showing the kinetics of the response and (e) the comparison of the maximum elevations in [Ca2+]cyt compared to the resting [Ca2+]cyt.

Supplementary Figure 6 HPLC purification and analysis by mass spectrometry of lipid A of P. aeruginosa H4.

(a) Reversed phase HPLC profile obtained from the LPS of P. aeruginosa H4 after mild acetate buffer hydrolysis. A semi-preparative Kromasil C18 column (5 µm, 100 Å, 20 x 250 mm, MZ Analysentechnik) was used and the sample (4 mg/ml in CHCl3/CH3OH 4:1, v/v) was eluted using a gradient that consisted of methanol-chloroform-water (57:12:31, v/v/v) containing 10 mM NH4OAc as mobile phase A and chloroform-methanol (70.2:29.8, v/v) with 50 mM NH4OAc as mobile phase B. The initial solvent system consisted of 2% B and was maintained for 40 min, followed by a linear three step gradient raising from 2 to 17% B (40-100 min), 17 to 27% B (100-170 min), and 27 to 80% B (170-314 min). The solvent was held at 80% B for 20 min, the column re-equilibrated in 20 min to 2% B and held there for additional 21 min before the next injection. The flow rate was 4 ml/min using a splitter between the evaporative light-scattering detector (Sedex model 75C ELSD, S.E.D.E.R.E., France) equipped with a low-flow nebulizer recording the chromatogram and the fraction collector (fractions in 1 min intervals). Nitrogen (purity 99.996%) was used as gas to nebulize the post column flow stream at 3.5 bar into the detector at 50 °C setting the photomultiplier gain to 11. The detector signal was transferred to the Gilson HPLC Chemstation (Trilution LC, version 2.1, Gilson) for detection and integration of the ELSD signal. The major lipid A containing pools are indicated as LA (1) and (2), respectively. (b) The corresponding mass spectra of the two lipid A containing HPLC pools. Electrospray Ionization Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FT-ICR-MS) was performed on a 7 Tesla APEX Qe instrument (Bruker Daltonics, USA). Samples were dissolved in a water/propan-2-ol/7 M triethylamine/acetic acid mixture (50:50:0.06:0.02, v/v/v/v) and spectra were acquired in the negative ion mode with nano-ESI using the Triversa Nanomate (Advion, USA) as ion source with a spray voltage set to −1.1 kV. Mass spectra were recorded in broad band mode and the mass scale was externally calibrated with glycolipids of known structure. Spectra were charge deconvoluted; given mass numbers refer to the monoisotopic mass of neutral molecules. Mass peaks representing proton adducts of lipid A species are assigned in bold style, the other assigned mass peaks are belonging to sodium adducts (Δ = +22 u) of these molecules. The major species (VI) in Pa H4 LA (1) could be assigned to a penta-acylated lipid A composed of 2 x GlcN, 2 x P, 4 x 12:0(OH), and 1 x 10:0(OH) (monoisotopic MWcalc: 1462.816 u). Minor molecule species with a third phosphate moiety ((VII), MWcalc: 1542.777 u) or with an additional Ara4N residue ((IX), MWcalc: 1593.869 u) are present as well. The major species in Pa H4 LA (2), molecule (V), could be assigned to a penta-acylated lipid A as well, but with one 12:0 instead of a 12:0(OH) (composition: 2 x GlcN, 2 x P, 3 x 12:0(OH), 1 x 12:0, and 1 x 10:0(OH); MWcalc: 1446.821 u). (XI) corresponds to (V) + P (MWcalc: 1526.782 u) and (VIII) to (V) + Ara4N (MWcalc: 1577.874 u), (X) corresponds to (VIII) – P (MWcalc: 1497.910 Da). Mainly in pool (1), minor occurring tetra-acylated lipid A variants could be identified as well ((III) = (V) - 10:0(OH) (MWcalc: 1276.696 u); (IV) = (VI) - 10:0(OH) (MWcalc: 1292.691 u); (I) (MWcalc: 1196.735 u) and (II) (MWcalc: 1212.730 u) are the mono-phosphorylated forms of (III) and (IV), respectively). (c) Maximum [Ca2+]cyt after treatment with the indicated LPS fragments (25 µg/ml) in Col‑0AEQ controls, lore‑1 or sd1‑29AEQ mutants and complementation lines (CL), with aequorin reconstituted with 10 µM coelenterazine‑h. Data are pooled from two experiments (mean and s.d. of n = 4, 4, 4, 6, 4; 4, 6, 4, 6, 4; 4, 6, 4, 6, 4; 4, 4, 4, 6, 4; 4, 6, 4, 6, 3; 4, 5, 4, 6, 3 seedlings per genotype and treatment (left to right)). LA (1) and LA (2), HPLC lipid A fractions (1) and (2); LPS-OH, hydrazine-treated LPS carrying only the two amide-bound fatty acids; core OS, isolated core oligosaccharide. Full version of Figure 6c.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 2 (PDF 1560 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ranf, S., Gisch, N., Schäffer, M. et al. A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nat Immunol 16, 426–433 (2015). https://doi.org/10.1038/ni.3124

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing