Symbiosis between soybean (Glycine max) and rhizobia is essential for efficient nitrogen fixation. Rhizobial effectors secreted through the type-III secretion system are key for mediating the interactions between plants and rhizobia, but the molecular mechanism remains largely unknown. Here, our genome-wide association study for nodule number identified G. max Nodule Number Locus 1 (GmNNL1), which encodes a new R protein. GmNNL1 directly interacts with the nodulation outer protein P (NopP) effector from Bradyrhizobium USDA110 to trigger immunity and inhibit nodulation through root hair infection. The insertion of a 179 bp short interspersed nuclear element (SINE)-like transposon into GmNNL1 leads to the loss of function of GmNNL1, enabling bradyrhizobia to successfully nodulate soybeans through the root hair infection route and enhancing nitrogen fixation. Our findings provide important insights into the coevolution of soybean–bradyrhizobia compatibility and offer a way to design new legume–rhizobia interactions for efficient symbiotic nitrogen fixation.
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The gene-sequencing data of NopP and the 16S rRNA in Bradyrhizobium were uploaded to the NCBI website, and a list of the accession numbers is provided in Supplementary Table 5. VCF files for SNPs and indels for GWAS are available from the corresponding authors on request. The sequence of Glyma.02G076900 can be downloaded from the Soybase website (https://www.soybase.org/sbt/). The nucleotide or amino acid sequences of NopP in Bradyrhizobium species were retrieved from various databases (NCBI, http://www.ncbi.nlm.nih.gov; Rhizobase, http://genome.annotation.jp/rhizobase/). Source data are provided with this paper.
Herridge, D. F., Peoples, M. B. & Boddey, R. M. Global inputs of biological nitrogen fixation in agricultural systems. Plant Soil 311, 1–18 (2008).
Wang, Z. & Tian, Z. Genomics progress will facilitate molecular breeding in soybean. Sci. China Life Sci. 58, 813–815 (2015).
Yang, S., Tang, F., Gao, M., Krishnan, H. B. & Zhu, H. R gene-controlled host specificity in the legume-rhizobia symbiosis. Proc. Natl Acad. Sci. USA 107, 18735–18740 (2010).
Indrasumunar, A. et al. Nodulation factor receptor kinase 1α controls nodule organ number in soybean (Glycine max L. Merr). Plant J. 65, 39–50 (2011).
Santos, M. A. et al. Mapping of QTLs associated with biological nitrogen fixation traits in soybean. Hereditas 150, 17–25 (2013).
Hwang, S. et al. Genetics and mapping of quantitative traits for nodule number, weight, and size in soybean (Glycine max L.[Merr.]). Euphytica 195, 419–434 (2014).
Oldroyd, G. E. Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat. Rev. Microbiol. 11, 252–263 (2013).
Haney, C. H. et al. Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3. Plant Cell 23, 2774–2787 (2011).
Mus, F. et al. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. Appl. Environ. Microbiol. 82, 3698–3710 (2016).
Ivanov, S., Fedorova, E. & Bisseling, T. Intracellular plant microbe associations: secretory pathways and the formation of perimicrobial compartments. Curr. Opin. Plant Biol. 13, 372–377 (2010).
Gourion, B., Berrabah, F., Ratet, P. & Stacey, G. Rhizobium-legume symbioses: the crucial role of plant immunity. Trends Plant Sci. 20, 186–194 (2015).
Zipfel, C. & Oldroyd, G. E. Plant signalling in symbiosis and immunity. Nature 543, 328–336 (2017).
Sugawara, M. et al. Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity. Nat. Commun. 9, 3139 (2018).
Kawaharada, Y. et al. Receptor-mediated exopolysaccharide perception controls bacterial infection. Nature 523, 308–312 (2015).
Yasuda, M. et al. Effector-triggered immunity determines host genotype-specific incompatibility in legume-Rhizobium symbiosis. Plant Cell Physiol. 57, 1791–1800 (2016).
Miwa, H. & Okazaki, S. How effectors promote beneficial interactions? Curr. Opin. Plant Biol. 38, 148–154 (2017).
Staehelin, C. & Krishnan, H. B. Nodulation outer proteins: double-edged swords of symbiotic rhizobia. Biochem. J. 470, 263–274 (2015).
Kereszt, A., Mergaert, P., Maroti, G. & Kondorosi, E. Innate immunity effectors and virulence factors in symbiosis. Curr. Opin. Microbiol. 14, 76–81 (2011).
Bartsev, A. V. et al. NopL, an effector protein of Rhizobium sp. NGR234, thwarts activation of plant defense reactions. Plant Physiol. 134, 871–879 (2004).
Ge, Y. Y. et al. The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate. J. Exp. Bot. 67, 2483–2494 (2016).
Zhang, L., Chen, X. J., Lu, H. B., Xie, Z. P. & Staehelin, C. Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling. J. Biol. Chem. 286, 32178–32187 (2011).
Ausmees, N. et al. Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234. J. Bacteriol. 186, 4774–4780 (2004).
Schechter, L. M., Guenther, J., Olcay, E. A., Jang, S. & Krishnan, H. B. Translocation of NopP by Sinorhizobium fredii USDA257 into Vigna unguiculata root nodules. Appl. Environ. Microbiol. 76, 3758–3761 (2010).
Skorpil, P. et al. NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii. Mol. Microbiol. 57, 1304–1317 (2005).
Ibanez, F., Wall, L. & Fabra, A. Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots. J. Exp. Bot. 68, 1905–1918 (2017).
Madsen, L. H. et al. The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. Nat. Commun. 1, 10 (2010).
Okazaki, S., Kaneko, T., Sato, S. & Saeki, K. Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system. Proc. Natl Acad. Sci. USA 110, 17131–17136 (2013).
Sprent, J. I. Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation. N. Phytol. 174, 11–25 (2007).
Schmutz, J. et al. Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183 (2010).
Zhou, Z. et al. Resequencing 302 wild and cultivated accessions identifies genes related to domestication and improvement in soybean. Nat. Biotechnol. 33, 408–414 (2015).
Glowacki, S., Macioszek, V. K. & Kononowicz, A. K. R proteins as fundamentals of plant innate immunity. Cell. Mol. Biol. Lett. 16, 1–24 (2011).
Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006).
Cui, H., Tsuda, K. & Parker, J. E. Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 66, 487–511 (2015).
Libault, M. et al. Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection. Plant Physiol. 152, 541–552 (2010).
Libault, M. et al. An integrated transcriptome atlas of the crop model Glycine max, and its use in comparative analyses in plants. Plant J. 63, 86–99 (2010).
Loh, J. T. et al. Population density-dependent regulation of the Bradyrhizobium japonicum nodulation genes. Mol. Microbiol. 42, 37–46 (2001).
Boogerd, F. C. & van Rossum, D. Nodulation of groundnut by Bradyrhizobium: a simple infection process by crack entry. FEMS Microbiol. Rev. 21, 5–27 (1997).
Xiao, T. T. et al. Fate map of Medicago truncatula root nodules. Development 141, 3517–3528 (2014).
Dodds, P. N. & Rathjen, J. P. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11, 539–548 (2010).
Maekawa, T., Kufer, T. A. & Schulze-Lefert, P. NLR functions in plant and animal immune systems: so far and yet so close. Nat. Immunol. 12, 817–826 (2011).
Yang, Y., Zhao, J., Morgan, R. L., Ma, W. & Jiang, T. Computational prediction of type III secreted proteins from Gram-negative bacteria. BMC Bioinform. 11, S47 (2010).
Batistic, O., Waadt, R., Steinhorst, L., Held, K. & Kudla, J. CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores. Plant J. 61, 211–222 (2010).
Fotiadis, C. T., Georgakopoulos, D. G., Dimou, M., Katinakis, P. & Tampakaki, A. P. Functional characterization of NopT1 and NopT2, two type III effectors of Bradyrhizobium japonicum. FEMS Microbiol. Lett. 327, 66–77 (2012).
Schauser, L., Roussis, A., Stiller, J. & Stougaard, J. A plant regulator controlling development of symbiotic root nodules. Nature 402, 191–195 (1999).
van de Sande, K. et al. Modification of phytohormone response by a peptide encoded by ENOD40 of legumes and a nonlegume. Science 273, 370–373 (1996).
Kang, Y. J. et al. Genome sequence of mungbean and insights into evolution within Vigna species. Nat. Commun. 5, 5443 (2014).
Li, Y. H. et al. De novo assembly of soybean wild relatives for pan-genome analysis of diversity and agronomic traits. Nat. Biotechnol. 32, 1045–1052 (2014).
Schmutz, J. et al. A reference genome for common bean and genome-wide analysis of dual domestications. Nat. Genet. 46, 707–713 (2014).
Gao, Y. et al. Out of water: the origin and early diversification of plant R-genes. Plant Physiol. 177, 82–89 (2018).
Vincent, J. M. in A Manual for the Practical Study of the Root-Nodule Bacteria. IBP Handbook no.15 (Blackwell Scientific Publications, Oxford Press, 1970).
Russell, D. W. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).
Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003).
Hood, E. E., Gelvin, S. B., Melchers, L. S. & Hoekema, A. New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218 (1993).
Chabaud, M. et al. in The Medicago truncatula Handbook pp. 1–8 (eds U Mathesius et al.) (The Samuel Roberts Noble Foundation, 2006); http://www.noble.org/MedicagoHandbook
Murray, M. G. & Thompson, W. F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4326 (1980).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Browning, B. L. & Browning, S. R. Genotype imputation with millions of reference samples. Am. J. Hum. Genet. 98, 116–126 (2016).
Browning, S. R. & Browning, B. L. Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering. Am. J. Hum. Genet. 81, 1084–1097 (2007).
Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012).
Yano, K. et al. Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice. Nat. Genet. 48, 927–934 (2016).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Felsenstein, J. PHYLIP (Phylogeny Inference Package) Version 3.696 (Univ. Washington, 2010).
Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874 (2016).
Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007).
Mather, K. A. et al. The extent of linkage disequilibrium in rice (Oryza sativa L.). Genetics 177, 2223–2232 (2007).
Hakoyama, T. et al. The SNARE protein SYP71 expressed in vascular tissues is involved in symbiotic nitrogen fixation in Lotus japonicus nodules. Plant Physiol. 160, 897–905 (2012).
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).
Lei, L. et al. A nodule-specific lipid transfer protein AsE246 participates in transport of plant-synthesized lipids to symbiosome membrane and is essential for nodule organogenesis in Chinese milk vetch. Plant Physiol. 164, 1045–1058 (2014).
Maekawa, T. et al. Polyubiquitin promoter-based binary vectors for overexpression and gene silencing in Lotus japonicus. Mol. Plant Microbe 21, 375–382 (2008).
Di, Y. H. et al. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean. Biochem. Biophys. Res. Commun. 519, 819–823 (2019).
Donaldson, P. A. & Simmonds, D. H. Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean. Plant Cell Rep. 19, 478–484 (2000).
Hu, R., Fan, C., Li, H., Zhang, Q. & Fu, Y.-F. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol. Biol. 10, 93 (2009).
Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405 (1987).
Xiao, T. T. et al. Fate map of Medicago truncatula root nodules. Development 141, 3517–3528 (2014).
Wang, C. et al. Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus. Plant Mol. Biol. 82, 97–111 (2013).
Zhang, M. et al. The MAP4 kinase SIK1 ensures robust extracellular ROS burst and antibacterial immunity in plants. Cell Host Microbe 24, 379–391 (2018).
Wang, H. et al. Dual role of BKI1 and 14-3-3 s in brassinosteroid signaling to link receptor with transcription factors. Dev. Cell 21, 825–834 (2011).
Yun, B. W. et al. S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature 478, 264–268 (2011).
We thank H. Liao (Fujian University of Agriculture and Forestry Science and Technology) and J. Zhao (Anhui Agriculture University) for providing some accessions of soybeans for the GWAS experiments and evolution analysis; G. Stacey (University of Missouri) for providing GUS-tagged USDA110; H. Liao (Fujian University of Agriculture and Forestry Science and Technology) for providing A. rhizogenes K599; and X. Yao (Huazhong Agriculture University) for providing the GV3101 strain with the ER marker gene OFP-HDEL. This work was supported by grant 2016YFD0100700 of The National Key Research and Development Program of China (to B.Z. and Y.L.) and 2015CB910200 of The National Key Basic Research Foundation of China (to X.W.), grants 31870257, 91535104 and 31430046 of The National Natural Science Foundation of China (to X.W.), grant 31471522 of The National Natural Science Foundation of China (to M.Z.) and grant CARS-004-PS06 of China Agriculture Research System (to C.Y.).
The authors declare no competing interests.
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Zhang, B., Wang, M., Sun, Y. et al. Glycine max NNL1 restricts symbiotic compatibility with widely distributed bradyrhizobia via root hair infection. Nat. Plants 7, 73–86 (2021). https://doi.org/10.1038/s41477-020-00832-7