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The BRUTUS iron sensor and E3 ligase facilitates soybean root nodulation by monoubiquitination of NSP1

Nature Plants volume 11, pages 595–611 (2025)Cite this article

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Abstract

Legumes form root nodules with symbiotic nitrogen-fixing rhizobacteria, which require ample iron to ensure symbiosis establishment and efficient nitrogen fixation. The functions and mechanisms of iron in nitrogen-fixing nodules are well established. However, the role of iron and the mechanisms by which legumes sense iron and incorporate this cue into nodulation signalling pathways remain unclear. Here we show that iron is a key driver of nodulation because symbiotic nodules cannot form without iron, even under conditions of sufficient light and low nitrogen. We further identify an iron optimum for soybean nodulation and the iron sensor BRUTUS A (BTSa) which acts as a hub for integrating iron and nodulation cues. BTSa is induced by rhizobia, binds to and is stabilized by iron. In turn, BTSa stabilizes and enhances the transcriptional activation activity of pro-nodulation transcription factor NSP1a by monoubiquitination from its RING domain and consequently activates nodulation signalling. Monoubiquitination of NSP1 by BTS is conserved in legumes to trigger nodulation under iron sufficiency. Thus, iron status is an essential cue to trigger nodulation and BTSa integrates cues from rhizobial infection and iron status to orchestrate host responses towards establishing symbiotic nitrogen fixation.

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Fig. 1: Iron levels determine root nodulation in soybean.
Fig. 2: NSP1a is involved in low-Fe-mediated inhibition of nodulation.
Fig. 3: BTSa interacts with NSP1a to promote soybean nodulation.
Fig. 4: BTSa responds to iron stress to regulate NSP1 stability and transcriptional activity.
Fig. 5: BTSa promotes NSP1a stability by monoubiquitination modification.
Fig. 6: Monoubiquitination of NSP1 by BTS is conserved in legumes.

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All data supporting the findings of this study are available in the Article and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

We thank Y. Liu from Huanan Agriculture University for providing the CRISPR–Cas9 vector. This work was supported by the National Key Research and Development Plan 2023YFD1200600 (to X.L.); National Natural Science Foundation of China grants 32090062 (to Z.W.), 3247150855 (to Z.W.) and 32330078 (to X.L.); Fundamental Research Funds for the Central Universities grant 2021ZKPY012 (to Z.W.) and X2662024ZKPY003 (to Z.W.); National Key Research and Development Program of China grant 2019YFA0904703 (to X.L.); and Jilin Scientific and Technological Development Program grant 20220402059GH (to L.Z.).

Author information

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China

    Ziyin Ren, Haizhen Li, Mi Yang, Xuesong Wu, Runxu Hu, Jingjing Lu, Hui Wang, Xinying Wu, Zhijuan Wang & Xia Li

  2. Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, China

    Ling Zhang

  3. Hubei Hongshan Laboratory, Wuhan, China

    Xia Li

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  1. Ziyin Ren
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  2. Ling Zhang
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Contributions

X.L. and Z.W. designed the experiments. Z.R., L.Z., H.L., M.Y., Xuesong Wu, R.H., J.L., H.W. and Xinying Wu performed the experiments. X.L., Z.W. and Z.R. wrote the manuscript.

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Correspondence to Zhijuan Wang or Xia Li.

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Nature Plants thanks Manuel Gonzalez-Guerrero, Peter Gresshoff, Penelope Smith and Takuya Suzaki for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Iron levels determine nodulation success in soybean.

a and d, Representative images (a) and number (d) of infection threads in wild-type Wm82 roots at 3 DAI. Scale bars = 200 µm. Data are the means ± SD of n = 10, 10, 12, 12 for 0, 0.4, 40, 400 µM Fe. b and e, Representative images (b) and number (e) of nodule primordia in Wm82 roots at 6 DAI. Scale bars = 1 mm. Data are the means ± SD of n = 12, 12, 10, 12 for 0, 0.4, 40, 400 µM Fe. c and f, Representative images of nodules (c) and Nodule numbers (f) at 18 DAI. Scale bars = 5 mm. Data are the means ± SD of n = 26, 17, 17, 19 for 0, 0.4, 40, 400 µM Fe. g, Phenotypes of soybean plants under different iron conditions. Plants were imaged at 21 DAI. Scale bars = 5 cm. h and i, DAB staining (h) and NBT staining (i) of roots at 1 DAI. Scale bar = 5 mm in (h) and 0.5 mm in (i). j, ROS content in roots at 1 DAI, Data are means ± SD of n = 3 (uninoculated 0.4 μM), n = 5 (inoculated 160 μM) and n = 4 (the others). k-n, Expression of NSP1a (k), NSP1b (l), NSP2a (m) and NSP2b (n) under different iron conditions at 1 DAI. Data are means ± SD of n = 3. For the box-plot in d, e and f, whiskers indicate minimum and maximum values, line indicates the median, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles. d-f, Significant differences were determined by one-way ANOVA (P < 0.05); j, k-n, significant differences were determined by two-way ANOVA (P < 0.05). The experiments were performed for three times with similar results.

Source data

Extended Data Fig. 2 Detection of native NSP1 by anti-NSP1 antibody and CRISPR–Cas9 gene editing of NSP1a.

a, Western blot of native NSP1a abundance in nodules of WT and nsp1a-1 mutant using anti-NSP1 antibody. The arrow indicates the specific band of NSP1a and NSP1b. The experiment was repeated for three times with similar results. b, Schematic of the NSP1a gene. The nsp1a-1 mutant in the Wm82 background has a 10-bp (540–549 bp) deletion in the coding region induced by CRISPR–Cas9. The red font in the mutant represents the new translational stop codon due to a frame shift arising from Cas9 nuclease activity. c, Diagram of wild-type and mutant NSP1a protein. d, Sanger sequencing chromatograms of wild-type Wm82 and nsp1a-1 mutant. The blue box indicates deleted region in the nsp1a-1 mutant.

Source data

Extended Data Fig. 3 Phylogenetic analysis of BTS orthologs and interaction assay between BTSb and NSP1a.

a, Phylogenetic tree and domain structures of select BTS orthologs. Clustal-W was used to align orthologs and homologs from Glycine max, Medicago truncatula, Lotus japonicus and Arabidopsis thaliana. MEGA-X was used to construct the phylogenetic tree with 1,000 bootstrap replications. b, Reference-free alignment of BTS proteins in Arabidopsis thaliana (At), Glycine max (Gm), Medicago truncatula (Mt) and Lotus japonicus (Lj) generated by TBtools. c, Yeast two-hybrid assay of the BTSb C-terminal domain (BTSb-CTer) and NSP1a. BD: Binding domain. AD: Activation domain. -LW: Leu and Trp dropout. -LWHA: Leu, Trp, His and Ade dropout. The experiment was repeated for three times with similar results.

Extended Data Fig. 4 BTSa and BTSb expression assay and CRISPR–Cas9 editing of BTSa and BTSb.

a, Expression profile of soybean BTSa and BTSb genes using data deposited on Phytozome. A heatmap was generated by TBtools. The color scale shows increasing expression levels from blue to red. b, RT–qPCR analysis of BTSa and BTSb in different tissues. Samples were collected at 21 DAI. Expression was normalized to ELF1b and values are means of three technical repetitions and SEM. The experiment was repeated for three times with similar results. c and d, Histochemical staining of GUS in the hairy roots with (+ R) or without (- R) inoculation (c) and nodules at different developmental stages (d) expressing BTSapro:GUS. Scale bars = 0.5 mm. The experiment was repeated for three times with similar results. e and f, CRISPR–Cas9 was used to mutate BTSa and BTSb using four sgRNAs targeting both BTSa and BTSb in exon 1 and 2. Two mutants were recovered (assigned btsa btsb-1 and btsa btsb-2) with edits in exon 2. Characterization of BTSa and BTSb edits in btsa btsb-1 (e) and btsa btsb-2 (f). Top: Schematic of the genomic structure of BTSa. Bottom: Alignment of BTSa, BTSb, BTSc and BTSd sequences in Wm82, btsa btsb-1 and btsa btsb-2. The sequence in blue represents the sgRNA, asterisks represent deletions detected in the edited allele, and the red font in mutants represent the stop codon arising from frame shifts induced by editing. g, Schematic of proteins encoded by Wm82 and the mutants.

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Extended Data Fig. 5 BTSa and BTSb play a similar role in regulating nodulation.

a and b, Nodulation phenotype (a) and nodule number (b) of hairy roots expressing BTSa or BTSb at 21 DAI. Scar bars = 2 cm. The numbers in the column of (b) represent the number of samples, data are the means ± SD and significant differences were determined by one-way ANOVA (P < 0.05). c, BTSa-MYC and BTSb-MYC expression in hairy roots in (a). Data are the means ± SD of n = 12. d, Characterization of BTSa editing in btsa-1 mutant. The red stars indicate deletion base pairs. e and f, Nodulation phenotypes (e) and nodule number (f) at 21 DAI. Scar bars = 2 cm for roots and 5 mm for nodules. Data are the means ± SD of n = 10, 11, 15 for WT, btsa-1, btsa btsb-2. g, Nitrogenase activity at 21 DAI. Data are the means ± SD of n = 9, 8, 8 for WT, btsa-1, btsa btsb-2. h, Characterization of BTSb editing in btsb-1 mutant. The red stars indicate the deletion base pairs. i and j, Nodulation phenotypes (i) and nodule number (j) at 21 DAI. Scar bars = 2 cm for roots and 5 mm for nodules. Data are the means ± SD of n = 16, 20, 13 for WT, btsa-1, btsa btsb-2. k, Nitrogenase activity of WT, btsb-1 and btsa btsb-2 at 21 DAI. Data are the means ± SD of n = 7, 8, 8 for WT, btsa-1, btsa btsb-2. f, g, j and k, Significant differences were determined by two-sided Student’s t-test (** P < 0.01, ***P < 0.001, ****P < 0.0001). For the box-plot in (f, g, j and k), whiskers indicate minimum and maximum values, line indicates the median, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles. The experiments were performed for three times with similar results.

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Extended Data Fig. 6 Amino-acid alignment of BTS proteins, detection of BTSa-MBP, stability assay of BTSa–MYC and BTSb–MYC and nodulation phenotypes of btsa btsb double mutants.

a, Amino-acid alignment of the BTS proteins from soybean, Lotus japonicus, Medicago truncatula and Arabidopsis thaliana. Hemerythrin (HHE) domains were marked with an orange underline, conserved histidine, glutamate and aspartate residues that potentially bind to metals are marked with red arrows. The RING zinc-finger domain was marked with a blue underline and the conserved cysteine and histidine are marked with blue asterisks. b, Coomassie Blue staining of proteins purified from E.Coli. The arrows indicate bands of MBP-HHE (with three HHE domains) and MBP-BTSa. c, In vivo stability of BTSa–MYC and BTSb–MYC under different iron conditions. The BTSa-MYC and BTSb-MYC fusion proteins were expressed in N. bethamiana-leaf and treated without CHX or with CHX plus 40 μM FAC or DFP. Anti-GFP was used as control of expression efficiency, anti-Actin or Ponceau S staining as loading controls. The numbers at the bottom denote the relative intensities normalized to BTSa-MYC intensity in the sample without CHX. FAC: Ferric ammonium citrate, DFP: deferiprone. The experiments were performed for three times with similar results. d, Nodulation phenotypes of WT and btsa btsb double mutants under different iron conditions. Number of nodules per plant were harvested and evaluated at 21 DAI. Scale bars = 2 mm. e, Nitrogenase activity of btsa btsb mutants under different iron conditions. Data are the means ± SD of n = 7 (WT in LFe, btsa btsb-1, 2 in NFe), 9 (WT in NFe, btsa btsb -2 in LFe) and 10 (btsa btsb-1 in LFe). Significant differences were determined by two-way ANOVA (P < 0.05). For the box-plot, whiskers indicate minimum and maximum values, line indicates the median, the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles. The experiments were performed for three times with similar results.

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Extended Data Fig. 7 Transcriptional and protein stability assays of NSP1a.

a, qRT–PCR analysis of NSP1a in uninoculated (-R) and inoculated (+R) roots of WT Wm82 and btsa btsb-1 grown under NFe and LFe conditions sampled at 1 DAI. Expression was normalized to ELF1b and values are means of three technical repetitions and SEM. The experiments were repeated for three times with similar results. Significant differences were determined by two-way ANOVA (P < 0.05). NFe: 10 mM Fe-citrite, LFe: 200 mM AA-DP. b, Cell-free degradation assay with recombinant GST–NSP1a in protein extracts from N. bethamiana leaves transiently transformed with UBIpro:BTSa–3×HA or an empty-vector control (HA). The samples were incubated for the indicated times and protein abundance was determined by western blotting. The numbers at the bottom denote the relative intensities of GST–NSP1a normalized to 0 point. The experiments were repeated for three times with similar results. c, Quantification of GST–NSP1a band intensities in (b). d, In vivo analysis of NSP1a–MYC stability in the presence or absence of BTSa–HA. UBIpro: NSP1a–4×MYC was transiently expressed in N. bethamiana leaves with UBIpro:BTSa–3×HA or vector control (HA-EV). The samples were collected 48 h after infiltration and prepared for western blotting. The numbers at the bottom denote the relative intensities of NSP1a–MYC normalized to respective HA-EV control. e, Quantification of NSP1a–MYC band intensities in (d). Data are the means ± SD of n = 4. Significant difference was determined by two-tailed Student’ t-test (**P < 0.01).

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Extended Data Fig. 8 BTSa possesses E3 ubiquitin-ligase activity assay of BTSa and characterization of ubiquitination sites in NSP1a by BTSa.

a and b, In vitro ubiquitination assay. BTSaRING (E3) fused to MBP was used with (+) or without (–) HA-tagged ubiquitin (3×HA–Ub), human E1 (UBE1) and E2 (UBE2D2). 500 ng MBP–BTSaRING recombinant protein and 500 ng 3×HA–Ubiquitin was loaded onto 10% SDS–PAGE gels and ubiquitinated proteins were detected by western blotting using an anti-HA antibody in (a) and anti-MBP antibody in (b). The experiment was repeated three times with similar results. c-g, Identification of NSP1a ubiquitination sites in vivo by LC–MS/MS. MS analysis was performed once. c, Schematic of NSP1a and the distribution of its ubiquitination sites. LCR: Low-complexity region. d-g, K133, K139, K143, K398 and K468 are potential ubiquitination sites.

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Extended Data Fig. 9 Monoubiquitination assays of NSP1b and NSP2a by BTSa, and complementary assays of NSP1a and NSP1a5KR in nsp1a-1 mutant.

a, Alignment of NSP1a, NSP1b, NSP2a and NSP2b. The red stars indicate the lysine monoubiqutinated by BTSa conserved in soybean, Medicago and Lotus, and the blue stars indicate the lysine conserved in soybean and Medicago or soybean and Lotus, b, In vivo ubiquitination assay of NSP1b by BTSa or AtBTS. Proteins extracted from N. bethamiana leaves transiently transformed with 35Spro:NSP1b–3×FLAG, 35Spro:UBQ–6×MYC, UBIpro:BTSa–3×HA or UBIpro:AtBTSa–3×HA. Anti-MYC agarose was used for immunoprecipitation and immunoprecipitated proteins were probed with anti-FLAG and anti-MYC antibodies. The experiments were repeated for three times with similar results. c, In vivo ubiquitination assay of NSP2a by BTSa or AtBTS. Proteins extracted from N. bethamiana leaves transiently transformed with 35Spro:NSP2a–3×FLAG, 35Spro:UBQ–6×MYC, UBIpro:BTSa–3×HA or UBIpro:AtBTSa–3×HA. Anti-MYC agarose was used for immunoprecipitation and immunoprecipitated proteins were probed with anti-FLAG and anti-MYC antibodies. The experiments were repeated for three times with similar results. d, Complementary assays of NSP1a and NSP1a5KR in nsp1a-1 mutant. Nodulation phenotype of hairy roots expressing NSP1apro:NSP1aWT–4×MYC (Npro:NSP1aWT) or NSP1apro:NSP1a5KR–4×MYC (Npro:NSP1a5KR) in Wm82 and nsp1a-1 mutant. The constructs contain a 35Spro:GFP expression cassette to visualize transgenic roots. + represents the transgenic roots with GFP fluorescence, – represents the non-transgenic roots without GFP fluorescence. Scar bars = 2 cm. The experiments were repeated for three times with similar results.

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Extended Data Fig. 10 Complementary assay of NSP1s and NSP13KR mutant, monoubiqutination specificity assay of NSP1a by BTSs.

a, Nodulation phenotype of transgenic soybean hairy roots in wild-type Wm82 and nsp1a-1 heterologously expressing UBIpro:MtNSP1WT–4×MYC, UBIpro:LjNSP1WT–4×MYC, UBIpro:MtNSP13KR–4×MYC or UBIpro:LjNSP13KR–4×MYC constructs. Nodules were harvested at 28 DAI. Scar bars = 2 cm. The experiments were repeated for three times with similar results. b, In vivo ubiquitination assay of NSP1a by AtBTS. Proteins extracted from tobacco leaves transiently transformed with (+) or without (–) 35Spro:NSP1a–3×FLAG, 35Spro:UBQ–6×MYC, UBIpro:BTSa–3×HA, UBIpro:AtBTSa–3×HA and empty vectors. Anti-MYC agarose was used for immunoprecipitation. The experiment was repeated for three times with similar results. c, Co-IP assay to test the interaction of AtBTSa–3×HA and NSP1a–3×FLAG or NSP2a–3×FLAG in N. benthamiana leaves. Protein was immunoprecipitated with HA antibody-bound agarose beads, immunoprecipitated proteins were analyzed using anti-FLAG and anti-HA antibodies. The experiments were repeated for three times with similar results.

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Supplementary Information

Supplementary Tables 1 and 2.

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Source Data Figs. 1–6 and Extended Data Figs. 1 and 4–7

Statistical source data for Figs. 1–6 and Extended Data Figs. 1 and 4–7.

Source Data Figs. 2–6, and Extended Data Figs. 2 and 6–10

Unprocessed western blots and gels for Figs. 2–6, and Extended Data Figs. 2 and 6–10.

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Ren, Z., Zhang, L., Li, H. et al. The BRUTUS iron sensor and E3 ligase facilitates soybean root nodulation by monoubiquitination of NSP1. Nat. Plants 11, 595–611 (2025). https://doi.org/10.1038/s41477-024-01896-5

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