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Receptor-mediated exopolysaccharide perception controls bacterial infection

Abstract

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume–rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant’s epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant–bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

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Figure 1: Phenotype of epr3 suppressor mutant.
Figure 2: Domain structure of EPR3 receptor protein.
Figure 3: EPS perception promotes infection thread development.
Figure 4: EPS and truncated EPS are perceived by the EPR3 receptor.
Figure 5: EPR3 is Nod-factor-induced and expressed in epidermal cells.
Figure 6: Model for two-step receptor-mediated recognition of compatible rhizobia.

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Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The Epr3 gene sequence has been deposited in GenBank under accession number AB506700.1.

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Acknowledgements

This work was supported by the Danish National Research Foundation grant no. DNRF79 and the ERC Advanced Grant 268523. We thank S. Bucholdt for technical help with Epr3 promoter-GUS fusions, T. Brock-Nannestad for technical assistance with FT-ICR mass spectrometry and J. Sullivan for comments on the manuscript. A.M. and R.W.C. were supported by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, US Department of Energy grant (DE-FG02-93ER20097) to the DOE Center for Plant and Microbial Complex Carbohydrates at the Complex Carbohydrate Research Center.

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Contributions

Y.K., identification of EMS and LORE1 epr3 mutations, characterization of the Epr3 gene, and phenotypic characterization; S.K., phenotypic characterization; M.W.N., identification of TILLING epr3 mutations, characterization of the Epr3 gene, complementation experiments; C.T.H. and M.B.T., biotin tagging of EPS and maltohexaose and affinity studies; K.G. and M.V., purification of EPR3 ectodomain and affinity studies; A.M., R.W.C. and C.T.H., purification and characterisation of monomeric EPS; N.S., screening for suppressor mutants and isolation of the epr3-10 allele; M.H.A., technical assistance; S.U.A., analysis of next-generation sequencing data; L.K., localization in N. benthamiana; K.J.J., S.T. and C.W.R. conceived experiments; M.B. and S.R. conceived and coordinated experiments; J.S. conceived experiments and coordinated. JS wrote the manuscript with input from Y.K., S.K., M.W.N., M.B., C.T.H., M.B.T., A.M., C.W.R. and S.R.

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Correspondence to J. Stougaard.

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Extended data figures and tables

Extended Data Figure 1 Genetic mapping and gene structure of Epr3.

a, Genetic map57,58 around the Epr3 locus. Positions of bacterial artificial chromosome (BAC) and transformation-competent artificial chromosome (TAC) clones and of closest markers together with the number of informative recombinations delimiting the Epr3 locus are indicated. b, Alignment of genomic and cDNA sequences defined 10 exons in Epr3 and a gene structure spanning 4,770 bp. The Epr3 gene is marked together with the exon–intron structure of Epr3 and positions of mutations.

Extended Data Figure 2 Domain structure of EPR3 and homologues proteins.

a, Alignment of LysM modules and CxC motifs from EPR3 and plant homologues. The cysteine substituted in epr3-3 and the conserved proline substituted in epr3-10 are marked. Asterisks indicate conserved amino acids. b, Alignment of the full-length EPR3 protein and homologous full-length proteins from Medicago (XP_003613165.1), soybean (XP_003517716.1, XP_003530632.1), tomato (XP_004242179.1), Ricinus (XP_002527912.1), Fragaria (XP_004300916.1) and Sorghum (XP_002455766.1). Predicted N-terminal signal peptides are marked and highlighted in yellow. For EPR3 a signal peptide of 21 amino acids is predicted by SignalP 4.1, assuming that the second of two methionines (arrow) that are eight amino acids apart is the correct N terminus. The three extracellular LysM domains are marked and highlighted in green. The putative transmembrane region is marked and highlighted in yellow. The classical kinase domains are marked I to XI and highlighted in blue.

Extended Data Figure 3 Quantification of transcript levels.

a, b, Relative expression of Epr3 (a) and Nin (b) transcript levels in Gifu, epr3-10 and epr3-11 mutants at 8 and 24 h post inoculation (h.p.i.) with R7A, R7AexoB and R7AexoU. ce, Relative expression of Nin, Nfya1 and Epr3 transcripts in Gifu, epr3-10 and epr3-11 and nfr1-1 roots treated with purified Nod factors. Values are from three biological (each consisting of ten plants) and three technical replicates. Bars show the corresponding upper and lower 95% confidence intervals.

Extended Data Figure 4 Localization of EPR3 protein and expression of Epr3.

ac, Membrane localization of EPR3. a, Representative image of EPR3–eYFP transiently expressed in N. benthamiana leaves shows co-localization with the plasma membrane dye FM 4-64 shown in b. An overlay is shown in c. d, e, pEpr3::GUS (d) and pNin::GUS (e) expression in roots 14 and 4 days, respectively, after inoculation with R7AexoB and R7AexoU. In d, n = 15, n = 26 and n = 10 for water, R7AexoB and R7AexoU, respectively; in e, n = 10. Scale bars: ac, 20 µm; d, e, 0.5 mm.

Extended Data Figure 5 Phylogeny of the individual LysM modules of all seventeen Lotus LysM receptor kinases.

Note that among these receptor kinases EPR3 stands out as the only protein where both the LysM2 and LysM3 modules do not cluster with the corresponding LysM2 and LysM3 modules of the other receptor kinases.

Extended Data Figure 6 Competition for infection following R7AexoU pre-inoculation.

a, Nodulation kinetics of Gifu inoculated with R7A (n = 30) or R7AexoB (n = 29). Nodule numbers represent infected nodules only. b, Time course of nodule numbers on Gifu, epr3-10 and epr3-11 inoculated with R7A and R7AexoB or pre-inoculated with R7AexoU followed by inoculation with R7A or R7AexoB 96 h later. n = 30, except for epr3-10 R7A (n = 20), epr3-10 R7AexoB (n = 19), epr3-10 R7AexoU/R7A (n = 28) and epr3-10 R7AexoU/R7AexoB (n = 28). c, Control experiment. Time course of nodule numbers on Gifu, epr3-10 and epr3-11 inoculated with R7A and R7AexoB or pre-inoculated with R7AΔnodA followed by inoculation with R7A or R7AexoB 96 h later. n = 30, except for epr3-10 R7A (n = 20) and epr3-10 R7AnodA/R7AexoB (n = 29). **P < 0.01 and *P < 0.05 (t-test) indicate significant differences in nodule number compared to groups at the same d.p.i. and indicated by matching coloured dots. Error bars show s.e.m.

Extended Data Figure 7 Purification of the EPR3 ectodomain.

a, EPR3 ectodomain expressed in insect cells was purified twice using Ni-IMAC affinity columns followed by a Superdex 75 10/300 GL column size exclusion. In the SEC profile shown, pure EPR3 ectodomain elutes as a single peak (Peak 2), at 12.2 ml elution volume. Contaminants elute in the void volume (V0; Peak 1). mAU is UV absorbance at 280 nm. b, Corresponding 15% Coomassie blue-stained SDS–PAGE gel. The EPR3 ectodomain from Peak 2 in different glycosylation states is visible as distinct bands (black arrow). Units on the right hand side list the molecular weight (kilodaltons) of marker proteins in the corresponding position in the first lane to the left.

Extended Data Figure 8 Isolation and characterization of EPS and the EPS–biotin conjugate by high-performance liquid chromatography and mass spectrometry.

a, Size-exclusion chromatography profile of monomeric EPS isolated from culture media of R7AΔndvB. R7AΔndvB is deprived of a 2-linked cyclic glucan24. The size-exclusion chromatography separation resolved one major fraction observed by refractive index detection, and the molecular size of the eluting fraction was assigned based on retention times of authentic standards of polysaccharides and MALDI–TOF mass spectrometry. The MALDI–TOF mass spectrometry analysis of the collected fraction was acquired in negative ionization mode and demonstrated the presence of a major monoisotopic [M-H] ion at m/z 1,437.39 (calculated [M-H] = 1,437.4055, and formula weight = 1,439.19) consistent with a Hex5GlcARibA octasaccharide substituted with three OAc groups. Vertical arrows mark column void volume and retention time of a 1,000 Da standard. b, Synthesis of EPS octaose–biotin conjugate. Conditions: (i) Aminooxy-thiol OEG linker, aniline, acetate buffer, pH 4.5, 40 °C, 16 h; (ii) triethylsilane, trifluoroacetic acid, 15 min; (iii) biotin–iodoacetamide reagent, borate buffer, pH 8.3, 15 min. c, High-performance liquid chromatography chromatogram at 206 nm. The chromatogram displays several overlapping peaks, owing to a microheterogenous distribution of OAc groups, ranging from 1 to 3. The inset shows the chromatogram for the maltohexaose–biotin conjugate. d, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum of EPS octaose–biotin conjugate. Inset displays isotopic distribution of the [M+H, 3Ac]+ ion. e, Structure and fragmentation pattern of EPS octaose–biotin conjugate. The displayed m/z values refer to d.

Extended Data Table 1 The number of infected nodules and uninfected nodules (‘bumps’) on wild-type Gifu and epr3 mutants 6 weeks after inoculation with R7AexoU DsRed
Extended Data Table 2 Infection of plant mutants.

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Kawaharada, Y., Kelly, S., Nielsen, M. et al. Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308–312 (2015). https://doi.org/10.1038/nature14611

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