Abstract
Legumes, unlike other plants, have the ability to establish symbiosis with nitrogen-fixing rhizobia. It has been theorized that a unique property of legume root cortical cells enabled the initial establishment of rhizobial symbiosis1,2,3. Here we show that a SHORTROOT–SCARECROW (SHR–SCR) stem cell program in cortical cells of the legume Medicago truncatula specifies their distinct fate. Regulatory elements drive the cortical expression of SCR, and stele-expressed SHR protein accumulates in cortical cells of M. truncatula but not Arabidopsis thaliana. The cortical SHR–SCR network is conserved across legume species, responds to rhizobial signals, and initiates legume-specific cortical cell division for de novo nodule organogenesis and accommodation of rhizobia. Ectopic activation of SHR and SCR in legumes is sufficient to induce root cortical cell division. Our work suggests that acquisition of the cortical SHR–SCR module enabled cell division coupled to rhizobial infection in legumes. We propose that this event was central to the evolution of rhizobial endosymbiosis.
This is a preview of subscription content, access via your institution
Relevant articles
Open Access articles citing this article.
-
A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation
Nature Communications Open Access 23 October 2023
-
Microbiome specificity and fluxes between two distant plant taxa in Iberian forests
Environmental Microbiome Open Access 22 July 2023
-
GmSPX8, a nodule-localized regulator confers nodule development and nitrogen fixation under phosphorus starvation in soybean
BMC Plant Biology Open Access 01 April 2022
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Data availability
All data generated or analysed during this study are included in this article and its Supplementary Information. Sequencing data for gene-expression analysis (RNA-seq) reported in this study have been deposited in the publicly available NCBI BioProject database under accession PRJNA599246, Sequence Read Archive accession numbers SAMN13741852–SAMN13741857. RNA-seq data for the 24 h and 120 h post S. meliloti spot inoculation were obtained from Schiessl et al.30 (Data S1). The M. truncatula Tnt1 transposon insertion lines used in this study were obtained from the Noble Foundation Tnt1 database (http://medicago-mutant.noble.org/mutant/database.php). The blot source data are provided in the Supplementary Information. Source data are provided with this paper.
Change history
11 January 2021
This Article was amended to correct the Peer review information, which was originally incorrect.
References
Geurts, R., Xiao, T. T. & Reinhold-Hurek, B. What does it take to evolve a nitrogen-fixing endosymbiosis? Trends Plant Sci. 21, 199–208 (2016).
Foucher, F. & Kondorosi, E. Cell cycle regulation in the course of nodule organogenesis. Plant Mol. Biol. 43, 229–242 (2000).
Gualtieri, G. & Biselling, T. The evolution of nodulation. Plant Mol. Biol. 42, 181–194 (2000).
Beijerinck, M. W. Rhizobial systematic. Botany Zeitung 46, 796–804 (1888).
Hellriegel, H. & Wilfarth, H. Untersuchungen Über die Stickstoffnahrung der Gramineen und Leguminosen (Buchdruckerei der ‘Post’ Kayssler) (1888).
Huisman, R. & Geurts, R. A roadmap toward engineered nitrogen-fixing nodule symbiosis. Plant Commun 1, 100019 (2020).
Oldroyd, G. E. D. & Downie, J. A. Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu. Rev. Plant Biol. 59, 519–546 (2008).
Timmers, A. C. J., Auriac, M. C. & Truchet, G. Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements. Development 126, 3617–3628 (1999).
Xiao, T. T. et al. Fate map of Medicago truncatula root nodules. Development 141, 3517–3528 (2014).
Truchet, G. et al. Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature 351, 670–673 (1991).
Gauthier-Coles, C., White, R. G. & Mathesius, U. Nodulating legumes are distinguished by a sensitivity to cytokinin in the root cortex leading to pseudonodule development. Front. Plant Sci 9, 1901 (2019).
Hirsch, A. M., Larue, T. A. & Doyle, J. Is the legume nodule a modified root or stem or an organ sui generis? Crit. Rev. Plant Sci. 16, 361–392 (1997).
Cooper, J. B. & Long, S. R. Morphogenetic rescue of Rhizobíum melíloti nodulation mutants by trans-Zeatin secretion. Plant Cell 6, 215–225 (1994).
Di Laurenzio, L. et al. The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86, 423–433 (1996).
Datta, N. & Cashmore, A. R. Binding of a pea nuclear protein to promoters of certain photoregulated genes is modulated by phosphorylation. Plant Cell 1, 1069–1077 (1989).
Gilmartin, P. M., Sarokin, L., Memelink, J. & Chua, N. H. Molecular light switches for plant genes. Plant Cell 2, 369–378 (1990).
Kucho, K., Yoshioka, S., Taniguchi, F., Ohyama, K. & Fukuzawa, H. Cis-acting elements and DNA-binding proteins involved in CO2-responsive transcriptional activation of Cah1 encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol. 133, 783–793 (2003).
Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E. & Benfey, P. N. Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development 127, 595–603 (2000).
Hiratsu, K., Matsui, K., Koyama, T. & Ohme-Takagi, M. Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739 (2003).
Vernié, T. et al. The NIN transcription factor coordinates diverse nodulation programs in different tissues of the Medicago truncatula root. Plant Cell 27, 3410–3424 (2015).
Long, Y. et al. SCARECROW-LIKE23 and SCARECROW jointly specify endodermal cell fate but distinctly control SHORT-ROOT movement. Plant J. 84, 773–784 (2015).
Cui, H. et al. An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants. Science 316, 421–425 (2007).
Helariutta, Y. et al. The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101, 555–567 (2000).
Gallagher, K. L., Paquette, A. J., Nakajima, K. & Benfey, P. N. Mechanisms regulating SHORT-ROOT intercellular movement. Curr. Biol. 14, 1847–1851 (2004).
Mathesius, U., Charon, C., Rolfe, B. G., Kondorosi, A. & Crespi, M. Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition. Mol. Plant Microbe Interact. 13, 617–628 (2000).
Tirichine, L. et al. A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis. Science 315, 104–107 (2007).
Murray, J. D. et al. A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis. Science 315, 101–104 (2007).
Yu, Q. et al. Cell-fate specification in Arabidopsis roots requires coordinative action of lineage instruction and positional reprogramming. Plant Physiol. 175, 816–827 (2017).
Henry, S. et al. SHR overexpression induces the formation of supernumerary cell layers with cortex cell identity in rice. Dev. Biol. 425, 1–7 (2017).
Schiessl, K. et al. NODULE INCEPTION recruits the lateral root developmental program for symbiotic nodule organogenesis in Medicago truncatula. Curr. Biol. 29, 3657–3668 (2019).
Soyano, T., Shimoda, Y., Kawaguchi, M. & Hayashi, M. A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus. Science 366, 1021–1023 (2019).
Soltis, D. E. et al. Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms. Proc. Natl Acad. Sci. USA 92, 2647–2651 (1995).
Griesmann, M. et al. Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361, eaat1743 (2018).
Gou, J. Y., Felippes, F. F., Liu, C. J., Weigel, D. & Wang, J. W. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23, 1512–1522 (2011).
Zhu, Q. et al. Development of “purple endosperm rice” by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system. Mol. Plant 10, 918–929 (2017).
Boisson-Dernier, A. et al. Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol. Plant Microbe Interact. 14, 695–700 (2001).
Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).
Cosson, V., Durand, P., d’Erfurth, I., Kondorosi, A. & Ratet, P. in Agrobacterium Protocols (ed. Wang, K.) (Humana, 2006).
Lauressergues, D. et al. The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2. Plant J. 72, 512–522 (2012).
Patel, R. K. & Jain, M. NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7, e30619 (2012).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protocols 7, 562–578 (2012).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Anders, S. & Huber, W. Differential Expression of RNA-Seq Data at the Gene Level—the DESeq Package (EMBL, 2013).
Kurihara, D., Mizuta, Y., Sato, Y. & Higashiyama, T. ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142, 4168–4179 (2015).
Coen, E. S. et al. floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell 63, 1311–1322 (1990).
Paciorek, T., Sauer, M., Balla, J., Wiśniewska, J. & Friml, J. Immunocytochemical technique for protein localization in sections of plant tissues. Nat. Protocols 1, 104–107 (2006).
Gietz, R. D. & Woods, R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002).
Chen, H. et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 146, 368–376 (2008).
Nozaki, M. et al. A missense mutation in the glucosamine-6-phosphate N-acetyltransferase-encoding gene causes temperature-dependent growth defects and ectopic lignin deposition in Arabidopsis. Plant Cell 24, 3366–3379 (2012).
Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).
Acknowledgements
We thank G. Oldroyd, J. Xu, X. Chen, and P. Benfey for suggestions on the manuscript; G. Oldroyd for lbd16-1 seeds; W. Cai for confocal imaging; and Life Science Editors for editing assistance. The research was supported by the Strategic Priority Research Program ‘Molecular Mechanism of Plant Growth and Development’ of the Chinese Academy of Sciences (XDB27040207), the National Science Foundation (31825003, 31730103, 32088102 to E. W., and 31500198), the National Key R&D Program of China (2019YFA0904703, 2016YFA0500502), the Ministry of Agriculture of China for Transgenic Research (2016ZX08009-003-001, 2016ZX08009003005-003), the Youth Innovation Promotion Association CAS (2016255) and 153D31KYSB20160074 (The Samuel Roberts Noble Foundation). The M. truncatula Tnt1 insertion mutant lines were obtained from The Samuel Roberts Noble Foundation and were created through research funded, in part, by a grant from the National Science Foundation, NSF-0703285.
Author information
Authors and Affiliations
Contributions
W.D. and E.W. directed the research. W.D., Y.Z., H.C., J.Y., J.S., J.G. and W.Y. performed most of the experiments. C.W., L.L., Y.W., X.Z., H.D., Y.M., L.X., Z.H., C.S., S.W. and N.Y. contributed to the analytical, molecular cloning, and transformation work. E.W. oversaw the entire study. W.D., D.W. and E.W. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature thanks Kimberly Gallagher, Jan Lohmann, Takashi Soyano and Krzysztof Szczyglowski for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Representative images of GUS staining of transformed hairy root tips in M. truncatula.
The Medicago orthologues of pivotal genes required for plant development in other species were identified by protein similarity (double-direction BLAST). The promoter length of these genes is indicated. Roots were stained in GUS staining solution about 60 min. These experiments were repeated two times with similar results. Scale bars, 100 μm. Details of these genes are in Supplementary Table 2.
Extended Data Fig. 2 Two regulatory elements in the MtSCR promoter govern its cortical cell expression in A. thaliana and cortical expression of SCR in other legumes.
a, AtSCR promoter-specific expression in QC and endodermis of stable transformed A. thaliana. Promoter truncation analysis in stable transformed A. thaliana shows that the AT1-box (−1,604 bp to −1,615 bp) and enhancer (−1,632 bp to −1,638 bp) elements of the MtSCR promoter are essential, and function synergistically to govern MtSCR expression in cortical and epidermal cells in A. thaliana. Cis-elements were predicated by using website PlantPAN 2.0 (http://plantpan2.itps.ncku.edu.tw/). C, cortex; E, endodermis; QC, quiescent centre. b, Sections of GUS-stained hairy roots show MtSCR and AtSCR expression patterns in M. truncatula. Transverse sections were made at about 3–5 mm from the root tip. c, In situ hybridization shows cortical expression of MtSCR in wild-type M. truncatula but not in Mtscr-1. d, Comparative genomics analysis reveals that legume and Parasponia SCR gene promoters harbour closely located (less than 100 bp) enhancer and AT1-box elements, whereas larger distances (170 bp or longer) between AT1-box and enhancer, or the absence of at least one of these elements, were observed in species outside the nitrogen fixation clade. e, In situ hybridization shows cortical cell expression of SCR in G. max, L. japonicus, C. arietinum, P. sativum and L. albus. f, PsSCR promoter (3,257 bp) and LaSCR2 promoter (3,162 bp) expressed in endodermis, QC and cortex of M. truncatula hairy root tips, whereas StSCR promoter (2,660 bp) shows endodermis-specific expression pattern. Note that LaSCR2 promoter (3,162 bp) showed very weak expression in Medicago transgenic hairy root. These experiments were repeated two times with similar results. C, cortex; E, endodermis; QC, quiescent centre. Scale bars, 20 μm (a, c, e, f) and 100 μm (b).
Extended Data Fig. 3 Root development and nodule formation in M. truncatula Mtscr mutants.
a, Schematic representation of MtSCR genomic sequence (top) and identification of Tnt1 insertion mutant of MtSCR (bottom). MtSCR genomic DNA is composed of 2 exons and 1 intron. Tnt1 insertion sites are indicated by arrows. b, Relative MtSCR expression levels in wild-type, Mtscr-1 and Mtscr-2 roots (n = 3). Expression levels were normalized against the reference gene EF-1. Data are mean ± s.d. c, d, Nodule numbers of wild-type, Mtscr-1 and Mtscr-2 roots (c) and representative images of plants and nodules (d) at 7 dpi, 14 dpi, 21 dpi and 28 dpi. e, Density of lateral root numbers (top) and nodules and primordia (bottom) in wild-type, Mtscr-1 and Mtscr-2 roots (n ≥ 20). These experiments were repeated three times with similar results. f, Nodulation phenotype of progeny from Mtscr-1 crossed with Mtscr-2 is comparable with their parents at 14 dpi, indicating that the Mtscr-1 mutation is allelic to the Mtscr-2 mutation (n ≥ 8). b, e, f, Two-sided Student’s t-test (*P < 0.05; **P < 0.01). c, Significance tests for nodule primordia (a/b/c/d) and nodules (a′/b′/c′/d′/e′) were performed. Conditions with different letters were significantly different (ANOVA, Duncan’s multiple range tests; P < 0.05). c, e, f, Boxes show the first quartile, median and third quartile; whiskers indicate minimum and maximum values. Data points are represented by dots. n represents independent biological samples. Scale bars, 1 cm (plants) and 1 mm (root nodules).
Extended Data Fig. 4 Developmental phenotype in Mtscr, Mtscr-1Mtscl23 and complementation plants.
a, Primary and lateral root radial patterning defects in Mtscr and Mtscr-1Mtscl23 mutants. Note that M. truncatula wild-type roots formed 4–5 layers of cortical cells whereas Mtscr roots formed only 3–4 layers of cortical-like cells. b, All of pMtSCR:MtSCR, pMtSCR∆En∆AT1:MtSCR and pAtSCR:MtSCR could complement Mtscr-1 hairy root radial patterning. c–e, Stable transgenic Mtscr-1 mutant transformed with pAtSCR:MtSCR shows normal shoot gravitropism (c) and root radial patterning (d), but shows a significant reduction in nodule formation at 14 dpi (e) (n ≥ 23). Letters (a/b) indicate significant differences (ANOVA Duncan’s multiple range tests; P < 0.01). e, Boxes show the first quartile, median and third quartile; whiskers indicate minimum and maximum values. f, Relative MtSCR expression levels in empty vector (WT), empty vector (Mtscr-1) and Mtscr-1pAtSCR:MtSCR stable transgenic lines (n = 6). Expression levels were normalized against the reference gene EF-1. Two-sided Student’s t-test (**P < 0.01). Data are mean ± s.d. In a, b, d, transverse sections were made at about 3–5 mm from the root tip. Red represents liginin deposition stained with phloroglucinol. White arrows in Mtscr-1, Mtscr-2, Mtscr-1Mtscl23 and empty vector (Mtscr-1) indicate lignin deposition between adjacent cells. Black arrows indicate endodermal cells, which are thin and long in wild-type or complementary hairy roots while in Mtscr-1, Mtscr-2 and Mtscr-1Mtscl23 mutant they are thick and round. P, pericycle; E, endodermis; m, mutant cell layer. Scale bars, 100 μm. n represents independent biological samples. Data points are represented by dots. These experiments were repeated three times with similar results. Lignin is produced in association with xylem differentiation and is deposited into the secondary cell walls of tracheary elements and fibre cells50.
Extended Data Fig. 5 SCR expression in cortical cells is required for nodulation symbiosis in M. truncatula, L. japonicus and G. max.
a, Nodulation phenotype at 21 dpi in wild-type (WT) hairy roots transformed with an empty vector (EV), pLjUBQ:MtSCR-SRDX, pMtSCR:MtSCR-SRDX or pMtNRT1.3:MtSCR-SRDX (n ≥ 21). b, Representative images show that hairy roots transformed with MtSCR-SRDX have greatly decreased nodule numbers compared with empty vector. White arrows indicate nodules. c, Hairy root lengths of M. truncatula at 21 dpi transformed with empty vector (EV), pLjUBQ:MtSCR-SRDX, pMtSCR:MtSCR-SRDX, or pMtNRT1.3:MtSCR-SRDX (n ≥ 21). d, Relative expression levels of MtSCR-SRDX in transgenic hairy roots from a. Expression levels were normalized against the reference gene EF-1. Data are mean ± s.d. e, Nodulation phenotype in L. japonicus hairy roots transformed with empty vector or pMtNRT1.3:LjSCR-SRDX at 21 dpi. (n ≥ 12). f, Representative images of empty vector and pMtNRT1.3:LjSCR-SRDX transgenic hairy roots. White arrows indicate nodules. g, Hairy root lengths of L. japonicus at 21 dpi transformed with empty vector and pMtNRT1.3:LjSCR-SRDX (n ≥ 12). h, Relative expression levels of LjSCR-SRDX in transgenic hairy roots from e. Expression levels were normalized against the reference gene LjUb. Data are mean ± s.d. i, Nodulation phenotype in G. max hairy roots transformed with an empty vector or pMtNRT1.3:GmSCR-SRDX at 21 dpi (n ≥ 35). j, Representative images of empty vector and pMtNRT1.3:GmSCR-SRDX transgenic hairy roots. White arrows indicate nodules. k, Hairy root lengths of G. max at 21 dpi transformed with empty vector and pMtNRT1.3:GmSCR-SRDX (n ≥ 35). l, Relative expression levels of GmSCR-SRDX in transgenic hairy roots from i. Expression levels were normalized against the reference gene GmELF1B. Data are mean ± s.d. a, c, Asterisks indicate significant differences from the empty vector control (ANOVA, Dunnett’s multiple tests; **P < 0.01; ns, not significant). e, g, i, k, Asterisks indicate significant differences by two-sided Student’s t-test (*P < 0.05; ns, not significant). a, c, e, g, i, k, Boxes show the first quartile, median and third quartile; whiskers indicate minimum and maximum values. n represents independent biological samples. Data points are represented by dots. These experiments were repeated three times with similar results. Scale bars, 1 mm.
Extended Data Fig. 6 MtSCL23 and MtSCR function redundantly in nodule organogenesis.
a, Maximum likelihood phylogenetic tree of SCR from A. thaliana; Oryza sativa (Q2RB59, A2ZAX5); Zea mays (NP_001168484); M. truncatula (Medtr7g074650); Selaginella moellendorffii (85562, 84762); Physcomitrella patens (Pp1s882_1V6.1, Pp1s85_139V6.1, Pp1s324_56V6.1) and its homologues from A. thaliana (AtSCL23, AtRGA1, AtSCL3, AtLAS/SCL18) and M. truncatula (Medtr4g076020, Medtr1g069725, Medtr3g065980, Medtr7g027190, Medtr8g442410). Branch support was obtained from 1,000 bootstrap repetitions. Evolutionary analyses were conducted in MEGA751. b, GUS staining of hairy roots transformed with pMtSCL23:EGFP-GUS or pMtSCL2:EGFP-GUS shows both MtSCL23 and MtSCL2 expressed in the cortical cells. Transverse sections were made at about 3–5 mm from the root tip. c, Mutant identification (left) and relative gene expression (right) levels of MtSCL23 (top) and MtSCL2 (bottom). Tnt1 insertion sites and qPCR primers are indicated. d, Nodulation phenotype of wild-type and Mtscl23 roots at 14 days after Sm1021 inoculation (n ≥ 24). e–g, Nodule numbers (e), lateral root numbers (f) and root lengths (g) of wild-type, Mtscr-1, Mtscl2, and Mtscr-1Mtscl2 roots at 14 dpi and 21 dpi (n ≥ 15). h, i, Primary root length (h) and lateral root numbers (i) of wild-type, Mtscr-1, and Mtscr-1Mtscl23 roots at 7 dpi, 14 dpi, 21 dpi and 28 dpi. Mtscr-1Mtscl23 shows short primary root length and similar lateral root numbers compared with Mtscr-1 (n ≥ 19). j, k, The density of lateral roots in Mtscr-1Mtscl23 plants is significantly increased compared with Mtscr-1 (j), whereas nodule density is significantly decreased (k) (n ≥ 19). l, Real-time RT–PCR (qPCR) analysis shows that induction of LBD16, ENOD40, FLOT4 and RIP upon Nod factor treatment for 24 h is fully dependent on MtSCR and MtSCL23. Expression of NIN, ERN1, NSP2 and VAPYRIN still can be induced, but the expression level was much lower than that in wild-type plants. Data are mean ± s.d. Expression levels were normalized against the reference gene EF-1. c–l, Two-sided Student’s t-test (*P < 0.05; **P < 0.01; ns, not significant). d–k, Boxes show the first quartile, median and third quartile; whiskers indicate minimum and maximum values. n represents independent biological samples. Data points are represented by dots. These experiments were repeated three times with similar results. Scale bars, 1 mm (b, root tip) and 50 μm (b, root sections).
Extended Data Fig. 7 MtSHR1 is expressed in the stele and MtSCR interacts with MtSHR1 and MtSHR2.
a, In situ hybridization shows that MtSHR1 is expressed in the stele. Scale bars, 100 μm. These experiments were repeated two times. b, Interactions between MtSCR and MtSHR1/2 in yeast two-hybrid assay. BD, binding domain; AD, activation domain. SD2, SD medium lacking leucine, tryptophan; SD4, SD medium lacking leucine, tryptophan, histidine and adenine. c, Split luciferase complementation indicates an in vivo interaction between MtSCR and MtSHR1/2 in N. benthamiana leaves. Fluorescence signal intensity is indicated. JW771 and JW772, which were derived from pCAMBIA2300 by insert Pro35S:nLUC or Pro35S:cLUC cassette, respectively, were used34. d, MtSCR co-immunoprecipitates with MtSHR1/2 in N. benthamiana. WB, western blot; IP, immunoprecipitation; CoIP, coimmunoprecipitation. MtSCR–HA and MtSHR1–FLAG or MtSHR2–FLAG were co-expressed in N. benthamiana leaves, respectively. For blot source data, see Supplementary Fig. 1. These experiments were repeated three times with similar results. e, Protein alignments show that two regions of SHR protein sequence show high variation.
Extended Data Fig. 8 M. truncatula MtSHR is required for nodule symbiosis.
a, Schematic representation of MtSHR2 genomic sequence (top) and identification of Tnt1 insertion mutant of MtSHR2 (bottom). b, Relative expression levels of MtSHR1 and MtSHR2 in Mtshr2 mutant (n ≥ 3). qPCR primer is indicated in a. c, Root length of wild-type and Mtshr2 roots at 14dpi (n ≥ 23). d, Numbers of nodules and primordia in wild-type and Mtshr2 roots at 14 dpi. e, Hairy root length of Mtshr2 at 21 dpi transformed with empty vector or MtSHR1 RNAi (n ≥ 16). f, Nodulation phenotype of Mtshr2 transformed with empty vector or MtSHR1 RNAi (n ≥ 16). g, qPCR showing that expression of MtSHR1 but not its homologues was significantly reduced in MtSHR1 RNAi transgenic hairy roots at 21 dpi (n ≥ 5). h, Representative images show that nodule numbers were reduced in MtSHR1-SRDX transgenic hairy roots of Mtshr2 plants. White arrows indicate nodules. i, Hairy root length of Mtshr2 at 21 dpi transformed with empty vector, pLjUBQ:MtSHR1-SRDX, pMtSHR1:MtSHR1-SRDX, or pMtNRT1.3:MtSHR1-SRDX (n ≥ 10). j, Relative expression levels of MtSHR1-SRDX in transgenic hairy roots in i. k, GUS staining shows that MtSHR1–SRDX-2NLS–GUS is specially located in cortical cells in pNRT1.3:MtSHR1-SRDX-2NLS-GUS transgenic hairy roots. l, Nodulation phenotype of Mtshr2 at 21 dpi transformed with empty vector or pMtNRT1.3:MtSHR1-SRDX-2NLS (n ≥ 10). m, Hairy root length of Mtshr2 plants at 21 dpi transformed with empty vector or pMtNRT1.3:MtSHR1-SRDX-2NLS. n, Relative expression levels of MtSHR1-SRDX-2NLS in transgenic hairy roots in m. In b, g, j and n, expression levels were normalized against the reference gene EF-1. Data are mean ± s.d. b–g, i, l, m, Two-sided Student’s t-test (*P < 0.05; **P < 0.01; ns, not significant). c–f, i, l, m, Boxes show first quartile, median and third quartile; whiskers indicate minimum and maximum values. n represents independent biological samples. Data points are represented by dots. These experiments were repeated three times with similar results. Scale bars, 1 mm (h) and 50 μm (k).
Extended Data Fig. 9 MtSHR1/2 and MtSCR are induced by cytokinins and required for NIN-induced spontaneous nodule formation, and overexpression of SHR promotes cortical cell division.
a, Immunoblotting showing that MtSHR1–GUS (~130 kDa) accumulated in pMtSHR1:MtSHR1-GUS transgenic hairy roots after 6-BA treatment for 4 days (n ≥ 10). For blot source data, see Supplementary Fig. 1. b, 6-BA treatment for 4 days upregulated the expression of MtSCR in root tips. Expression levels were normalized against the reference gene EF-1 (n ≥ 20). c, d, The frequency (c) and numbers (d) of spontaneous nodules induced at 10 weeks by transformation with pLjUBQ:NIN were significantly lower in Mtscr-1 than in wild-type plants (n ≥ 26). e, Section of spontaneous nodules formed in wild-type and Mtscr-1 roots, in the absence of S. meliloti in c. f, In A. thaliana, cortical cell divisions were evident within 24 h after initiating conditional ectopic overexpression (pG1090-XVE:AtSHR stable transformed line) of AtSHR via oestradiol treatment (10 μM). Arrowheads indicate cortical cell divisions. En, endodermis; Co, cortex; Ep, epidermis. These experiments were repeated two times with similar results. g, Relative expression levels of MtSCR and MtSHR1 in stable transformed rice (n = 6). Expression levels were normalized against the reference gene Cyclophilin2. h, Root sections of rice roots transformed with empty vector or overexpression of MtSCR-MtSHR1 lines. These experiments were repeated two times with similar results. i, Cortex-specific overexpression of MtSHR1 driven by the MtNRT1.3 promoter is sufficient to trigger cortical cell division. Transverse sections were made at about 3–5 mm from the root tip. These experiments were repeated three times with similar results. j, Table showing that excessive cortical cell division induced by MtSHR1 overexpression requires MtSCR, but does not require the Nod factor signalling pathway components NFP, NSP1, NSP2 and NIN. d, Boxes show first quartile, median and third quartile; whiskers indicate minimum and maximum values. b, d, g, Two-sided Student’s t-test (**P < 0.01). Data are mean ± s.d. n represents independent biological samples. Data points are represented by dots. Scale bars, 1 mm (c), 20 μm (f), 50 μm (e) and 100 μm (h, i).
Extended Data Fig. 10 The MtSHR–MtSCR module is required for infection thread formation and can be activated by symbiotic signals.
a, Histochemical analysis of ENOD11:GUS expression of pLjUBQ:MtSHR1-transformed roots. b, A higher frequency of pMtENOD11:GUS staining sites was observed in MtSHR1-overexpressing hairy roots compared with the empty vector control at 7 dpi with Sm1021. c, Average numbers of infection threads (ITs) and infection foci in wild-type, Mtscr-1, and Mtscr-1Mtscl23 roots 7 days after inoculation with LacZ-marked Sm1021 (n ≥ 12). Different letters (a/b) indicate significant differences (ANOVA, Duncan’s multiple range tests; P < 0.05). d, Average numbers of infection threads and infection foci in empty vector and pLjUBQ:MtSHR1-SRDX transformed hairy roots 7 days after inoculation with LacZ-marked Sm1021 (n ≥ 14). ITs includes infection threads that end in epidermis and reach cortex. e, Real-time RT–PCR (qPCR) analysis showing that expression of MtSCR but not MtSHR1/2 is induced in wild-type plants at 7 dpi (n = 5). f, GUS staining of hairy roots shows that pMtSCR:EGFP-GUS and pMtSHR1:MtSHR1-GUS but not pMtSHR1:EGFP-GUS are induced in nodule primordia. These experiments were repeated three times with similar results. g, qPCR showing that genetic impairment of MtSHR1/2 function significantly reduces the expression of MtSCR at 7 dpi (n ≥ 5). h, MtSCR induction in M. truncatula requires the Nod factor signalling pathway components NSP1, NSP2 and NIN (n ≥ 5). i, GUS protein is not modified and accumulated upon Sm1021 inoculation in p35S:GUS transgenic root. Actin is a loading control. These experiments were repeated two times with similar results. For blot source data, see Supplementary Fig. 1. j, Elevated levels of MtSHR1–GUS fusion proteins upon Sm1021 inoculation depend on the Nod factor signalling pathway components NSP1 and NSP2. These experiments were repeated three times with similar results. For blot source data, see Supplementary Fig. 1. k, qPCR shows that overexpression of MtSHR1 triggers upregulation of MtSCR (n = 5). l, ChIP–PCR shows that MtSHR1 associated with the promoter of an MtSCR fragment that does not overlap with the AT1-box (−1,604 bp to −1,615 bp) and enhancer (−1,632 bp to −1,638 bp). GFP–3×FLAG-transformed hairy roots were used as control. p35S:MtSHR1-3×FLAG or p35S:GFP-3×FLAG-transformed hairy roots were collected 21 days after Sm1021 inoculation. Fold-enrichment calculations from three replicate qPCR assays. These experiments were repeated in two independent ChIP experiments with similar results. m, n, The induction of LBD16 upon Nod factor treatment for 24 h was abolished in Mtscr-1Mtscl23 (m) and SHR1 dominant-negative transgenic roots (n). o, The induction of MtSCR upon Sm1021 treatment was abolished in lbd16-1 roots. p, MtSHR–MtSCR and LBD16 may regulate each other to form a positive feedback loop to coordinately regulate nodule organogenesis. b, d, e, g, h, k–o, Two-sided Student’s t-test (*P < 0.05; **P < 0.01; ns, not significant). e, g, h, k–o, Expression levels were normalized against the reference gene EF-1. b, Boxes show first quartile, median and third quartile; whiskers indicate minimum and maximum values. n represents independent biological samples. Data are mean ± s.d. Data points are represented by dots. These experiments were repeated three times with similar results. Scale bars, 1 mm (a) and 100 μm (f).
Extended Data Fig. 11 A proposed model of cortical cell division competence for nodule organogenesis in legumes.
a, The MtSHR–MtSCR network in cortical cells allows them to respond to symbiotic signals and cytokinins. b, Rhizobial signals activate the MtSHR–MtSCR module to promote cortical cell division and nodule organogenesis. c, SHR and SCR are not expressed in root cortical cells of A. thaliana. d, Ectopic expression of SHR–SCR network leads to cortical cell division in A. thaliana. Ep, epidermis; Co, cortex; En, endodermis; Vasc, vascular tissue; Lrc, lateral root cap; Col, columella.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1: Source data for images for blots. Original source images for all data obtained by western blots; Supplementary Table 2: Genes used for screening in this study; Supplementary Table 3: Promoter screening primers; Supplementary Table 4: Primers used in this study.
Supplementary Table
Supplementary Table 1: Differentially expressed genes in M. truncatula hairy roots overexpressing MtSHR1 (OE).
Rights and permissions
About this article
Cite this article
Dong, W., Zhu, Y., Chang, H. et al. An SHR–SCR module specifies legume cortical cell fate to enable nodulation. Nature 589, 586–590 (2021). https://doi.org/10.1038/s41586-020-3016-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-020-3016-z
This article is cited by
-
Microbiome specificity and fluxes between two distant plant taxa in Iberian forests
Environmental Microbiome (2023)
-
Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago
Nature Plants (2023)
-
A transcriptional activator effector of Ustilago maydis regulates hyperplasia in maize during pathogen-induced tumor formation
Nature Communications (2023)
-
Development of transgenic composite Stylosanthes plants to study root growth regulated by a β-expansin gene, SgEXPB1, under phosphorus deficiency
Plant Cell Reports (2023)
-
GmSPX8, a nodule-localized regulator confers nodule development and nitrogen fixation under phosphorus starvation in soybean
BMC Plant Biology (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
