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A soybean cyst nematode suppresses microbial plant symbionts using a lipochitooligosaccharide-hydrolysing enzyme

Abstract

Cyst nematodes are the most damaging species of plant-parasitic nematodes. They antagonize the colonization of beneficial microbial symbionts that are important for nutrient acquisition of plants. The molecular mechanism of the antagonism, however, remains elusive. Here, through biochemical combined with structural analysis, we reveal that Heterodera glycines, the most notorious soybean cyst nematode, suppresses symbiosis by secreting an enzyme named HgCht2 to hydrolyse the key symbiotic signalling molecules, lipochitooligosaccharides (LCOs). We solved the three-dimensional structures of apo HgCht2, as well as its chitooligosaccharide-bound and LCO-bound forms. These structures elucidated the substrate binding and hydrolysing mechanism of the enzyme. We designed an HgCht2 inhibitor, 1516b, which successfully suppresses the antagonism of cyst nematodes towards nitrogen-fixing rhizobia and phosphorus-absorbing arbuscular mycorrhizal symbioses. As HgCht2 is phylogenetically conserved across all cyst nematodes, our study revealed a molecular mechanism by which parasitic cyst nematodes antagonize the establishment of microbial symbiosis and provided a small-molecule solution.

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Fig. 1: H. glycines inoculation antagonizes rhizobium nodulation.
Fig. 2: Hydrolytic mode of HgCht2 towards different types of LCOs.
Fig. 3: Crystal structures of HgCht2 in the apo form and in complex with substrates.
Fig. 4: HgCht2 hampers LCO-induced symbiosis.

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Data availability

The mass spectrometry secretomic data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD051385. The RNA-sequencing data used in our analysis can be found in the Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA1099893. The atomic coordinates for the apo HgCht2, (GlcNAc)5-bound HgCht2 and SmNF-V-bound HgCht2 have been deposited in the Protein Data Bank (www.rcsb.org) with accession codes 8HW6, 8HW7 and 8HW8, respectively. Any other relevant data are available from the corresponding authors on request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Wang (Nanjing Agricultural University, China) for suggestions on the soybean hairy root experiment. We thank C. Staehelin (Sun Yat-sen University, China) for his collaborations in obtaining the NFs from S. meliloti strain 1021 (pEK327) at the very beginning of the project. We thank Q. Chen for his involvement in protein expression, purification and crystallization of HgCht2. We thank Y. Chen and J. Wu (Huazhong Agricultural University, China) for assistance in phenotyping experiments. We thank G. Stacey (University of Missouri, USA) for critical reading of the paper and valuable suggestions. We thank the staff of BL18U/BL19U1 Beamline of National Facility for Protein Science Shanghai at Shanghai Synchrotron Radiation Facility for assistance during data collection. This study was supported by the National Natural Science Foundation of China (32161133010, 32322072 and 32272595), the National Key Research and Development Program of China (2022YFD1700200), the Youth Innovation Program of Chinese Academy of Agricultural Sciences (Y2023QC03), the Innovation Program of Chinese Academy of Agricultural Sciences, the Shenzhen Science and Technology Program (KQTD20180411143628272) and the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District (PT202101-02).

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Contributions

Q.Y. conceived and designed this project in consultation with X.G. Q.Y. and W.C. provided funding. W.C. and D.W. conducted the structural, biochemical and physiological experiments. Q.Y. and W.C. analysed the structural data. X.G. and Y.C. supervised the experiments on rhizobium and nematode. S.K. conducted the synthesis of inhibitors. Q.Y. and W.X. designed the chemical inhibition experiments. Q.Y., W.C., D.W. and X.G. wrote the paper.

Corresponding authors

Correspondence to Xiaoli Guo or Qing Yang.

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Nature Microbiology thanks Kasper Andersen, Deepak Haarith, Peter Mergaert 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

Extended Data Table 1 List of the identified proteins from secretions of H. glycines when exposed to soybean roots
Extended Data Table 2 The chitinases in the transcriptome of ppJ2s of H. glycines
Extended Data Table 3 The dissociation constants of different molecules to HgCht2
Extended Data Table 4 X-ray data collection and structure-refinement statistics
Extended Data Table 5 Sequences for primers used in this study

Extended Data Fig. 1 The function, expression and sequence conservation of HgCht2.

a, Nodulation symptoms in soybean roots inoculated with dsRNA (GFP or HgCht2)-treated H. glycines. The uninoculated soybean plant served as the control. Bar = 3 mm. b, qRT-PCR analysis of the transcript level of HgCht2 after RNAi treatment. HgGAPDH was used as the internal control. Data are means ± SE (n = 3 independent experiments). c, SDS-PAGE (left panel) and hydrolytic activity (right panel) analysis of the recombinant HgCht2 and its mutant. Data are shown as mean ± SE (n = 3 independent experiments). d, Sequence alignment of cyst nematode chitinases in the same clade with HgCht2. e, Sequence alignment of HgCht2 and other three soybean cyst nematode chitinases. Asterisks in b, c represent statistically significant differences (Student’s two-sided unpaired t-test, ****P < 0.0001, ***P < 0.001). The black triangles in d, e represent the residues constituting the substrate-binding subsites of HgCht2.

Source data

Extended Data Fig. 2 The hydrolytic products of HgCht2 toward different substrates.

a, The hydrolytic mode of HgCht2 toward two Nod factors from Sinorhizobium meliloti 1021 (pEK327), SmNF-IV-Ac (C16:2, Ac, S) and SmNF-IV (C16:2, S). b–d, The structure and mass spectrum identification of BjNF-V-Ac (C18:1, Ac, MeFuc) (b) and its hydrolytic products, BjNF-III-Ac (C18:1, Ac) (c), BjNF-II-Ac (C18:1, Ac) (d). e–l, The structure and mass spectrum identification of SmNF-V (C16:2, S) (e), SmNF-IV-Ac (C16:2, Ac, S) (j), SmNF-IV (C16:2, S) (k), and their hydrolytic products, SmNF-III (C16:2) (f), SmNF-II (C16:2) (g), (GlcNAc)2-SO3H (h), (GlcNAc)3-SO3H (i), and SmNF-II-Ac (C16:2, Ac) (l). The red groups represent the substituents at the ends of the chitooligosaccharides backbone. The black arrows represent the [M-H]- ions or the formic acid adduct [M + HCOO]- ions of LCOs and the hydrolytic products.

Extended Data Fig. 3 The predicted structures and hydrolytic activities of other chitinases.

a, The predicted binding mode of SmNF-V (C16:2, S). The structures of cyst nematodes chitinases in the same clade as HgCht2, are predicted by AlphaFold. Superimposition of these predicted structures with the crystal structure of SmNF-V (C16:2, S)-bound HgCht2 indicates that these chitinases can accommodate the aliphatic chain and sulfonic acid group of SmNF-V (C16:2, S) (the green lines). SmNF-V (C16:2, S) is shown as blue sticks. The residues constituting the substrate-binding cleft are shown as yellow sticks. b, Western blots of the recombinantly expressed chitinases. c, Diagram showing the four residues of HgCht2 that potentially interact with the acyl group of SmNF-V (left panel), and the hydrolytic activity of HgCht2Q31A/I33A/E272A/Q337A toward SmNF-V (right panel). Data are means ± SE (n = 3 independent experiments). d, The enzymatic activities of different chitinases toward (GlcNAc)5 and SmNF-V (right panel). Data are means ± SE (n = 3 independent experiments). e, The hydrolytic activity of HgCht2 toward different substrates. Data are means ± SE (n = 3 independent experiments).

Source data

Extended Data Fig. 4 The enzymatic activity of HgCht2 is important for H. glycines-mediated suppression of rhizobia nodulation and mycorrhizal fungal symbiosis.

a, Nodules on hairy roots expressing HgCht2D129A/E131A or HgCht2 at 14 dpi. Hairy roots expressing GFP are used the control. Bar = 3 mm. b, Number of infection threads in hairy roots expressing HgCht2D129A/E131A and HgCht2 at 3 dpi. Data are shown as means ± SE (n = 5 biologically independent samples). c, qRT-PCR analysis of HgCht2 expression in hairy roots expressing HgCht2D129A/E131A or HgCht2 at 14 dpi. Data are shown as means ± SE (n = 3 independent experiments). d, Nodulation symptoms of soybean roots at 14 dpi upon treatment with HgCht2D129A/E131A or HgCht2. Bar = 3 mm. e, Nodule number of soybean roots at 14 dpi following treatment with HgCht2D129A/E131A or HgCht2. Soybean roots treated with PBS buffer were used as the control. Data are shown as means ± SE (n = 14 biologically independent samples) of one representative experiment. The experiment was repeated three times with similar results. f, Nodulation symptoms of soybean roots at 14 dpi with B. diazoefficiens under the HgCht2 treatment with or without 1516b. Bar = 5 mm. g, Infective thread formation in soybean roots at 3 dpi treated with HgCht2 and 1516b. White arrow, infected thread. Bar = 10 μm. h, Transcript expression levels of the early symbiosis marker genes GmENOD40 and GmNIN at 3 dpi under treatment with HgCht2 and 1516b. Data are shown as means ± SE (n = 3 independent experiments). i, Nodules of soybean roots at 14 dpi when treated with HgCht2 in the presence or absence of 1516b. Bar = 3 mm. j, Mycorrhizal fungal structures in soybean roots are shown at 7 dpi with R. irregularis in the presence of HgCht2 with or without 1516b. Bar = 100 μm. Soybean roots treated with PBS buffer were used as controls. Asterisks in b, c, e, h represent statistically significant differences (Student’s two-sided unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Source data

Extended Data Fig. 5 The synthesis, inhibitory activity and predicted binding mode of the target molecule 1516b.

a, Flow chart of 1516b synthesis. b, c, 1H NMR (b) and 13C NMR (c) spectrum for compound 1516b. d, The inhibitory activity of 1516b (100 µM) toward HgCht2 by using different substrates. Data are means ± SE (n = 3 independent experiments). e, The inhibitory activity of 1516b (100 µM) against different enzymes was evaluated using SmNF-V (C16:2, S) as the substrate. 1516b was dissolved in DMSO, and reactions with an equal volume of DMSO were used as the control. Data are means ± SE (n = 3 independent experiments). f, Surface representations of HgCht2 complexed with 1516b (left), and the interactions between 1516b and HgCht2 (right) is shown. The black dashed lines represent the H-bonds. The 1516b compound is shown as yellow sticks, and residues composing the 1516b binding site are labelled and shown as blue sticks. g, Superposition of the binding modes of 1516b (yellow) and SmNF-V (C16:2, S) (blue) in the HgCht2.

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Supplementary Data 1

The secretome data of pre-parasitic second-stage juveniles under stimulation with soybean roots and the transcriptome data of H. glycines.

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Chen, W., Wang, D., Ke, S. et al. A soybean cyst nematode suppresses microbial plant symbionts using a lipochitooligosaccharide-hydrolysing enzyme. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01727-5

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