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Plant receptor-like protein activation by a microbial glycoside hydrolase

Nature volume 610, pages 335–342 (2022)Cite this article

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Abstract

Plants rely on cell-surface-localized pattern recognition receptors to detect pathogen- or host-derived danger signals and trigger an immune response1,2,3,4,5,6. Receptor-like proteins (RLPs) with a leucine-rich repeat (LRR) ectodomain constitute a subgroup of pattern recognition receptors and play a critical role in plant immunity1,2,3. Mechanisms underlying ligand recognition and activation of LRR-RLPs remain elusive. Here we report a crystal structure of the LRR-RLP RXEG1 from Nicotiana benthamiana that recognizes XEG1 xyloglucanase from the pathogen Phytophthora sojae. The structure reveals that specific XEG1 recognition is predominantly mediated by an amino-terminal and a carboxy-terminal loop-out region (RXEG1(ID)) of RXEG1. The two loops bind to the active-site groove of XEG1, inhibiting its enzymatic activity and suppressing Phytophthora infection of N. benthamiana. Binding of XEG1 promotes association of RXEG1(LRR) with the LRR-type co-receptor BAK1 through RXEG1(ID) and the last four conserved LRRs to trigger RXEG1-mediated immune responses. Comparison of the structures of apo-RXEG1(LRR), XEG1–RXEG1(LRR) and XEG1–BAK1–RXEG1(LRR) shows that binding of XEG1 induces conformational changes in the N-terminal region of RXEG1(ID) and enhances structural flexibility of the BAK1-associating regions of RXEG1(LRR). These changes allow fold switching of RXEG1(ID) for recruitment of BAK1(LRR). Our data reveal a conserved mechanism of ligand-induced heterodimerization of an LRR-RLP with BAK1 and suggest a dual function for the LRR-RLP in plant immunity.

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Fig. 1: Specific recognition of XEG1 by RXEG1(LRR).
Fig. 2: Cryo-EM structure of the XEG1–RXEG1(LRR)–BAK1(LRR) complex.
Fig. 3: Mutagenesis of the XEG1–RXEG1(LRR)–BAK1(LRR) complex.
Fig. 4: RXEG1 inhibits the glycoside hydrolysis and virulence activities of XEG1.
Fig. 5: Mechanism of XEG1-induced RXEG1 activation.

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Data availability

The atomic coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics PDB and the Electron Microscopy Data Bank. The PDB codes for the XEG1–RXEG1(LRR)–BAK1(LRR), XEG1–RXEG1(LRR) (cryo-EM), BAK1(LRR) and RXEG1(LRR) structures are 7DRC, 7W3V, 7W3T and 7W3X, respectively. The Electron Microscopy Data Bank codes for the XEG1–RXEG1(LRR)–BAK1(LRR), XEG1–RXEG1(LRR), BAK1(LRR) and RXEG1(LRR) structures are EMD-30826, EMD-32294, EMD-32293 and EMD-32295, respectively. The PDB code for the XEG1–RXEG1(LRR) crystal structure is 7DRB. The filtered clean RNA-seq reads have been deposited in the National Center for Biotechnology Information database under the accession number GSE201521. Source data are provided with this paper.

Code availability

No custom code or mathematical algorithms were used in our study.

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Acknowledgements

We thank J.-M. Zhou from the Chinese Academy of Science for providing anti-BAK1 antibody, M. Zhang from Shaanxi Normal University for assistance in bacterial infection assays and M. Joosten for providing seeds of the SOBIR1/SOBIR1-sobir1/sobir1-like knockout mutant. This project was supported by the National Natural Science Foundation of China (numbers 32171193 and 31971119 to Z.H., number 31421001 to J.C., number 31721004 to Yuanchao Wang and numbers 32172423 and 31872927 to Yan Wang), the the China Agriculture Research System (number CARS-004-PS14 to Yuanchao Wang), the Alexander von Humboldt Foundation (Humboldt Professorship to J.C.), the Max-Planck-Gesellschaft (Max Planck Fellowship to J.C.), Deutsche Forschungsgemeinschaft SFB-1403-414786233 (J.C.), Germany’s Excellence Strategy CEPLAS (EXC-2048/1, Project 390686111; J.C.) and the National Key Research and Development Program of China (number 2021YFA1300701 to Z.H. and Yan Wang).

Author information

Author notes

  1. These authors contributed equally: Yue Sun, Yan Wang, Xiaoxiao Zhang

Authors and Affiliations

  1. Tsinghua-Peking Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Yue Sun, Xiaoxiao Zhang, Yu Xiao, Zhifu Han & Jijie Chai

  2. Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China

    Yan Wang, Zhaodan Chen, Yeqiang Xia, Lei Wang, Yujing Sun, Mingmei Zhang & Yuanchao Wang

  3. The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, China

    Yan Wang, Zhaodan Chen, Yeqiang Xia, Lei Wang, Yujing Sun, Mingmei Zhang & Yuanchao Wang

  4. Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Jijie Chai

  5. Institute of Biochemistry, University of Cologne, Cologne, Germany

    Jijie Chai

  6. Cluster of Excellence in Plant Sciences (CEPLAS), Düsseldorf, Germany

    Jijie Chai

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Contributions

J.C., Yuanchao Wang, Yan Wang and Z.H. conceived and conceptualized the study and designed the experiments. Yue Sun performed expression screening, purified the proteins and prepared the cryo-EM samples. X.Z. collected and analysed the cryo-EM data. Yu Xiao analysed the X-ray data. Yan Wang, Z.C., Yeqiang Xia, Yujing Sun and M.Z. performed the mutation, protein interaction, enzyme activity, plant transformation and infection assays and analysed the data. L.W. performed RNA-seq analyses. J.C., Yuanchao Wang, Yan Wang and Z.H. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Yan Wang, Zhifu Han, Yuanchao Wang or Jijie Chai.

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Extended data figures and tables

Extended Data Fig. 1 RXEG1LRR-XEG1 complex assembly and specific interaction mode.

a, Gel filtration analysis of XEG1 interaction with RXEG1LRR (in vitro complex). Top: gel filtration profiles of XEG1 (blue), RXEG1LRR (red) and XEG1 + RXEG1LRR (black). Right: SDS-PAGE analyses of fractions of the samples after gel filtration visualized Coomassie Blue staining. The experiment was performed three times independently with similar results. b, Gel filtration analysis of the co-expressed XEG1-RXEG1LRR complex. Shown on the left is the gel filtration profile and on the right is SDS-PAGE analysis of the peak fractions containing the complex. The experiment was performed three times independently with similar results. c, Structural alignment of XEG1 with FI-CMCase (PDB code: 5GM3). d, XEG1 surface charge distribution. e, Analyses of RXEG1 interaction with XEG1, FI-CMCase and Phytophthora elicitin INF1 by Co-IP. C-terminally eGFP-tagged RXEG1 was co-expressed with C-terminally HA-tagged XEG1, FI-CMCase or INF1 in N. benthamiana. Proteins were isolated 2 days after agroinfiltration, immunoprecipitated with GFP-trap A beads, and subjected to western blotting using anti-GFP or anti-HA. INF1 was used as a negative control. Experiments were repeated three times with similar results. f, Cell death triggered by XEG1, FI-CMCase and INF1 in N. benthamiana. C-terminally HA-tagged XEG1, FI-CMCase and INF1 were transiently expressed in N. benthamiana and cell death was photographed 3 days after agroinfiltrations (n ≥ 8 biologically independent samples). Experiments were repeated three times with similar results. Uncropped gels are shown in Supplementary Fig. 2.

Extended Data Fig. 2 Density maps of representative regions.

a, b, Detailed interactions between RXEG1LRR and XEG1. RXEG1ID, RXEG1N-loopout, XEG1 and RXEG1LRR are shown in cyan, blue, pink and light green, respectively. Densities are generated in PyMOL and contoured at 6 sigma. c,d, Detailed interactions between RXEG1LRR and BAK1LRR. RXEG1ID, BAK1LRR and RXEG1LRR are shown in cyan, purple and light green, respectively. Green mesh lines show electron density of residues involved in the interactions. Densities are generated in PyMOL and contoured at 6 sigma. e, Alignment of the crystal and cryo-EM structures of XEG1-RXEG1LRR. f, Detailed interactions between RXEG1ID and RXEG1LRR. Shown in g–i are the models and cryo-EM segments from different domains of the XEG1-RXEG1LRR-BAK1LRR complex: g, RXEG1ID (cyan), RXEG1N-loopout (blue) and the last four LRRs (light green), h, XEG1 (pink), i, BAK1LRR (purple). Densities are generated in PyMOL and contoured at 8 sigma.

Extended Data Fig. 3 RXEG1 mediates recognition of XEG1 and its homologues of GH12 proteins in N. benthamiana.

a, The editing type of RXEG1 in rxeg1 N. benthamiana genome. b, Interactions between RXEG1 with GH12 proteins derived from Phytophthora parasitica (P. parasitica), Phytophthora sojae (P. sojae) and Phytophthora infestans (P. infestans). C-terminally eGFP-tagged RXEG1 was co-expressed with HA-tagged XEG1 or its homologues in N. benthamiana indicated. Proteins were isolated 2 days after agroinfiltration, immunoprecipitated with GFP-trap A beads, and subjected to western blotting using anti-GFP or anti-HA. Experiments were repeated three times independently with similar results. c, Cell death triggered by XEG1 and its homologues in N. benthamiana depends on RXEG1. HA-tagged XEG1 and homologues were transiently expressed in wild type (WT) or rxeg1-knockout N. benthamiana. The Phytophthora elicitor INF1 that does not rely on RXEG1 for recognition was used as control. Cell death was monitored 3 days after agroinfiltrations (n ≥ 12 biologically independent samples). Experiments were repeated twice with similar results. d, The editing type of BAK1 in bak1 N. benthamiana genome. e, Morphological phenotypes of WT, rxeg1 and bak1 N. benthamiana. Five-week-old plants were photographed. f, Interactions between XEG1 with RXEG1 or its mutants. C-terminally HA-tagged XEG1 was co-expressed with eGFP-tagged RXEG1 and indicated mutants in N. benthamiana. Proteins were isolated 2 days after agroinfiltration, immunoprecipitated with GFP-trap A beads, and subjected to western blotting using anti-GFP or anti-HA. Experiments were repeated three times with similar results. g, RXEG1 mutants failed to rescue the XEG1-induced cell death phenotype in rxeg1 plants. C-terminally HA-tagged XEG1 was co-expressed with eGFP-tagged RXEG1 and indicated mutants in N. benthamiana. Cell death was monitored 3 days after agroinfiltrations (n ≥ 8 biologically independent samples). Experiments were repeated twice with similar results. h, Heat map of the expression of the genes responsive to XEG1 in wild-type (WT) N. benthamiana and indicated mutants. Differentially expressed genes were identified with a q-value < 0.05 and log2(fold change, FC) > 1 or < −1 relative to EV-treatment. i, Principal component analysis (PCA) of transcriptome in WT and indicated mutants treated with EV or XEG1 with BAM files colored based on two biological replicates. Uncropped gels are shown in Supplementary Fig. 2.

Extended Data Fig. 4 Sequence alignment of XEG1 and GH12 proteins.

Amino acid sequence alignment among GH12 family proteins. The first five proteins including XEG1 have previously been shown to interact with RXEG1. The residues from XEG1 interacting with RXEG1N-loopout or RXEG1ID are highlighted with green squares at the bottom. Sequence alignment was performed using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Extended Data Fig. 5 Cryo-EM reconstruction of the XEG1-RXEG1LRR and XEG1-RXEG1LRR-BAK1LRR complexes.

a, Representative cryo-EM micrograph of the XEG1-RXEG1LRR-BAK1LRR complex. b, Representative views of 2D class averages of the XEG1-RXEG1LRR-BAK1LRR complex. c, The cryo-EM image processing workflow. d, FSC curves at 0.143 of the final reconstruction of the XEG1-RXEG1LRR-BAK1LRR complex unmasked (red) or masked (black) and FSC curves at 0.5 for model refined against the final map (black), the first half map (blue) and the second half map (red). e, FSC curves at 0.143 and 0.5 of the final reconstruction of BAK1LRR. f, FSC curves at 0.143 and 0.5 of the final reconstruction of the XEG1-RXEG1LRR complex.

Extended Data Fig. 6 Expression of RXEG1∆CT confers Phytophthora resistance in N. benthamiana.

a, The level of transgene expression in the indicated rxeg1 N. benthamiana transformants assayed with semi-quantitative RT-PCR. Total RNA was isolated from leaves of four-week-old transgenic plants and used for cDNA synthesis. Expression levels of transgenes were determined with semi-quantitative RT-PCR using N. benthamiana EF-1α as an endogenous control. The fragments (RXEG1ΔCT-eGFP) amplified from the transgenes were shown in the schematic diagram of the constructs expressing RXEG1syn∆CT or derived mutants. b, Protein expression in the indicated rxeg1 N. benthamiana transformants. Proteins were isolated from leaves of six-week-old transformants with indicated transgene driven by RXEG1 native promoter, immunoprecipitated with GFP-trap A beads, and subjected to western blotting using anti-GFP. The amount of loading was quantified by anti-UGPase. Experiments were repeated twice with similar results. c, Disease symptoms in rxeg1 N. benthamiana transformants after inoculation with P. parasitica. Disease symptoms in each indicated tranformants were photographed 2 days after inoculation with P. parasitica zoospores. d, Overexpression of RXEG1syn∆CT suppresses Phytophthora parasitica infection in rxeg1 N. benthamiana. rxeg1 N. benthamiana transformants expressing RXEG1syn∆CT, the two indicated mutants or eGFP under the control of 35S promoter were inoculated with P. parasitica zoospores. Colonization of P. parasitica was quantified using genomic DNA by qPCR. Inoculated leaves were collected at 2 days post inoculation and used for DNA isolation. Colonization of P. parasitica was quantified using genomic DNA by qPCR and normalized to eGFP control which was set as 1. Data are presented as mean value ± SEM (n = 3 biologically independent replicates). Data were analyzed by one-way ANOVA followed by Dunnett’s test. e,f, Bacterial colonization in rxeg1 N. benthamiana transformants. Leaves of the indicated transformants were infiltrated with Pseudomonas syringae pv. tomato DC3000 HopQ1-1 mutant (DC3000ΔhopQ1) (OD = 0.0002). Bacteria titer was determined 2 and 3 days after inoculation. CFU, colony-forming units. Data are shown as box plots, with individual data points plotted (n = 8 biologically independent samples), The centre line, box edges and whiskers indicates the median, lower and upper quartiles and the minima and maxima data points, respectively. Data were analyzed by two-way ANOVA followed by Turkey’s test. Experiments were repeated three times with similar results. g, BAK1 was co-expressed with C-terminally eGFP-tagged RXEG1 in wild type and SOBIR1/SOBIR-like knockout N. benthamiana. Infiltrated leaves were collected 40 h after agroinfiltration upon treatment with purified XEG1 (1 µM) protein for 10 min. Proteins were immunoprecipitated with GFP-trap A beads, and subjected to western blotting using anti-GFP or anti-BAK1. Experiments were repeated three times with similar results. Uncropped gels are shown in Supplementary Fig. 2.

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Extended Data Fig. 7 Cryo-EM reconstruction of the apo-RXEG1LRR.

a, Representative cryo-EM micrograph of the apo-RXEG1LRR. b, Representative views of 2D class averages of the apo-RXEG1LRR. c, The cryo-EM image processing workflow. d, FSC curves at 0.143 of the final reconstruction of apo-RXEG1LRR unmasked (gray) or masked (black). e, FSC curves at 0.5 for model refined against the final map (black), the first half map (red) and the second half map (blue). f, The final EM density map of apo-RXEG1LRR, color coded to show the local resolution estimated by Relion.

Extended Data Fig. 8 Sequence alignment of LRR-RLPs from different plant species.

Amino acid sequence alignment of RXEG1 and some representative RLPs. The two loop regions RXEG1N-loopout and RXEG1ID are indicated by blue and cyan bars underneath, respectively. BAK1 interacting residues of RXEG1 around the RXEG1LRR-BAK1LRR interfaces are highlighted with blue squares at the bottom. The green 1, 2 and 3 at the bottom represents the disulfide bond formed by cysteine. Sequence alignment was performed using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Extended Data Table 1 X-ray diffraction data collection and refinement statistics
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Extended Data Table 2 Cryo-EM data collection, refinement and validation statistics
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Sun, Y., Wang, Y., Zhang, X. et al. Plant receptor-like protein activation by a microbial glycoside hydrolase. Nature 610, 335–342 (2022). https://doi.org/10.1038/s41586-022-05214-x

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