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Early stages of legume–rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins

Nature Plants volume 7, pages 428–436 (2021)Cite this article

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Abstract

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.

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Fig. 1: MtABCG56 is a root- and nodule-specific gene expressed during symbiosis.
Fig. 2: MtABCG56 is a PM cytokinin exporter.
Fig. 3: MtABCG56 affects the cytokinin signalling pathway and cytokinin homeostasis.
Fig. 4: MtABCG56 modulates nodulation efficiency.

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Data availability

The sequence data from this article can be found in Phytozome v.12.1 database under the following accession numbers: MtABCG56 (Medtr7g098320), MtABCG37 (Medtr7g098750), MtABCG39 (Medtr7g098760), MtABCG57 (Medtr7g098370), MtLOG3 (Medtr1g057020), MtRR4 (Medtr5g036480) and MtENOD11 (Medtr3g415670). Transcriptomic data were retrieved from refs. 1,8,20,24,25,26,50. M. truncatula R108 Tnt1 transposon insertion lines, namely NF19946 and NF11085, were obtained from the Samuel Roberts Noble Foundation. Source data are provided with this paper.

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Acknowledgements

We thank F. Frugier for mtcre1-1 seeds; the Noble Research Institute for mtabcg56 Tnt1 insertion mutant seeds; D. Weijers for the pPLV11_v02 binary vector; S. Bensmihen for the pDONR-ProLeEXT1 vector; A. van Zeijl for E. meliloti 2011/pMH682; B. G. Rolfe for E. meliloti 1021/pHC60 strain; M. J. Barnett for E. meliloti A2101/pE65 strain; and S. R. Long for E. meliloti Rm1021/pXLGD4. We thank E. Martinoia and J. Murray for critical comments; L. Charrier and Jie Liu for excellent technical assistance; and H. Martínková and I. Petřík for technical assistance with cytokinin profiling. This work was supported by the Polish National Science Centre (grants nos. 2015/19/B/NZ9/03548 and 2011/03/B/NZ1/02840). O.N. was supported by the Czech Science Foundation (grant no. 20-26232S) and M.G. by Swiss National Funds (project nos. 31003A_165877 and 310030_197563).

Author information

Authors and Affiliations

  1. Department of Plant Molecular Physiology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

    Karolina Jarzyniak, Joanna Banasiak, Tomasz Jamruszka, Aleksandra Pawela & Michał Jasiński

  2. Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Poznań, Poland

    Karolina Jarzyniak & Michał Jasiński

  3. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Martin Di Donato & Markus Geisler

  4. Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, The Czech Academy of Sciences, Olomouc, Czech Republic

    Ondřej Novák

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Contributions

M.J. devised and supervised the project. K.J. and M.J. designed the experiments and interpreted the results. K.J. performed the majority of the experiments (qRT–PCR analyses, promoter analyses, genotyping of mutants, phenotypic characterization of mutants and silenced material) and generated plasmids. J.B. generated non-tissue-specific silencing construct. A.P. generated and tested ProMtRR4 and ProABCG37,39,57 GUS fusion constructs. J.B. identified Tnt1 transposon insertion lines, assessed infection events, made the cross-section of the root/nodules, and performed light and confocal microscopy. A.P., T.J. and J.B. contributed to qRT–PCR analyses. T.J. prepared Nod factor. M.D.D. and M.G. designed and performed transport experiments and localized protein in Arabidopsis roots and N. benthamiana leaf epidermal cells. O.N. conducted quantification of endogenous cytokinins. K.J. conducted most of the statistical analyses. K.J. and J.B. prepared figures. K.J., M.G. and M.J. wrote the manuscript with the help of the co-authors. All authors saw and commented on the manuscript.

Corresponding author

Correspondence to Michał Jasiński.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Florian Frugier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 MtABCG56 is a NF-induced marker gene.

Expression of MtABCG56 (blue) and its close MtABCG homologues detected in recent transcriptomic experiments. Values represent the non-log transformed fold changes (FC) in expression of selected MtABCGs after Nod factor treatment (NF) and/or E. meliloti inoculation relative to control. Red color indicates statistically significant upregulation adopted from the following transcriptomic experiments. NOD 4 h – gene expression observed only 4 h after Nod Factor (NF) treatment; n/a – no data available; RH – isolated root hairs; LCM– laser-dissected epidermal cells; ART – all root tissues from root segments. 1 – RH with 4 h and 20 h 10-8 M NF treatment respectively (P < 0.05)50; 2 – RH with 24 h 10-6 M NF treatment and E. meliloti inoculated root hairs 5 days after inoculation, in the hyperinfected sickle (skl) mutant, affected in the Ethylene Insensitive 2 (EIN2) gene (P < 0.05 and FC > 2)25; 3 – LCM with 4 h and 24 h 10−8 M NF treatment respectively (P < 0.05 and FC > 2)1; 4 –10-9 M NF-treated root segments in the presence of 1 µM of aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis (3 h) (P < 0.001 and FC > 2)20; 5 –10-8 M NF-treated root segments (6 h and 24 h) (P < 0.05 and FC > 1.5)26; 6 – 10-7 M NF-treated root segments (10 h) (P < 0.05 and FC > 1.68)24.

Extended Data Fig. 2 Tissue-specific expression patterns of MtLOG3.

Transgenic roots carrying ProMtLOG3:NLS-tdTomato construct were fixed and observed under confocal microscopy after 4 h of NF treatment. Mock-treated roots are presented in the left panel. Upper panel in each section represents optical cross-section. Images are representative of n > 15 roots obtained from two independent experiments (transformations). tdTomato was pseudocoloured in red. Scale bars, 100 µm.

Extended Data Fig. 3 Expression pattern of MtABCG56 during different stages of the symbiotic interaction.

Composite transgenic plants carrying ProMtABCG56:GUS construct were imaged at different stages after inoculation with E. meliloti (E.m) Rm1021/pXLGD4 (lacZ). Double staining using Magenta-Gal and X-Gluc allowed the visualization of the infecting E. meliloti in magenta and MtABCG56 expression in blue. All images represent roots observed 4-14 dai. Scale bars, 100 µm.

Extended Data Fig. 4 Expression of MtABCG56 and its close MtABCG homologues in M. truncatula nodule zones.

The heat map represents the normalized RNAseq reads of MtABCG56 (blue) and its close MtABCG homologues in five nodule zones with a gradient from green (minimal expression) to magenta (maximal expression). Data from Roux et al.8, FI - Meristematic Zone; FIId - Infection Zone - distal fraction; FIIp - Infection Zone - proximal fraction; IZ - Interzone; ZIII – Nitrogen-fixing Zone.

Extended Data Fig. 5 Identification of mtabcg56 mutants.

a, The MtABCG56 gene consists of 24 exons. The positions of Tnt1 retrotransposon insertions in MtABCG56 are indicated by triangles (mtabcg56-2, 1st intron; mtabcg56-1, 10th exon). Blue and red arrows indicate location of primers used for genotyping of MtABCG56 WT and mutant alleles, respectively. b-c, MtABCG56 relative expression in corresponding WT, mtabcg56-1, complemented mtabcg56-1 (b) and mtabcg56-2 (c) roots. d, MtABCG56 relative expression in WT and mtabcg56-1 transformed with empty vector, and mtabcg56-1 roots carrying complementation constructs under epidermal-specific (ProLeEXT1) or cortex-specific (ProCO2) promoters. The data represent the mean ± s.d. of 6 biological replicates obtained from two independent experiments. e-f, Shoot and root dry weights (DWs) and root length of wild type, corresponding WT (white boxes), mtabcg56-1 (e), and mtabcg56-2 (f) (grey boxes), 21 days after germination. For each box-and-whiskers plot: the centre black line represents the median; a ‘+’ represents the mean; the box extends from the 25th to 75th percentiles; the whiskers are drawn down to the 10th percentile and up to the 90th. Points below and above the whiskers are drawn as individual dots. N represents the number of individual plants obtained from two independent experiments. Identical or different letters indicate no or significant differences, respectively, P < 0.05 (b, d, e, f); P < 0.01 (c). P values, determined by the Kruskal-Wallis test with a post hoc Dunn’s multiple comparison test (b, d, e, f) and two-tailed Mann-Whitney test (c), can be found in Supplementary Data. e-f, Images of representative wild type R108, corresponding WT, mtabcg56-1 (e), and mtabcg56-2 (f), 21 days after germination.

Source data

Extended Data Fig. 6 Relative expression of MtABCG56 and its close homologues in roots carrying MtABCG56 silencing constructs under various promoters.

a,b, Relative expression of MtABCG56 and its close homologous in roots carrying MtABCG56 silencing construct under epidermal-specific (ProLeEXT1-RNAi56), cortex-specific (ProCO2-RNAi56) (a) as well as CaMV 35 S (b) promoters and empty vector as a control, 6 h after inoculation with the E. meliloti E65 strain (closed circles) or in mock-treated roots (open circles). For transgenic hairy roots, the actin gene and the dsRED gene (transformation control) were used as references. The data represent the means ± s.d. of 6 biological replicates collected from different composite plants. Identical or different letters indicate no or significant differences (P < 0.05), respectively. P values, determined by two-way ANOVA with a post hoc Tamhane’s T2 multiple comparison test (a, b), can be found in Supplementary Data.

Source data

Extended Data Fig. 7 mtabcg56 is less sensitive to CK.

a,b, Root length of R108 WT, corresponding WT and abcg56 mutants measured 14 days after transfer on BAP 10−8 M. The data represent the means ± s.d. Different letters represent significant differences (P < 0.05). P values, determined by the Kruskal-Wallis test with a post hoc Dunn’s multiple comparison test, can be found in Supplementary Data. N represents the number of individual plants.

Source data

Extended Data Fig. 8 MtABCG56 modulates nodulation efficiency.

a,b, Shoot and root dry weights (DWs), root length, and nodule numbers per shoot and root DWs, and root length of wild type R108 and corresponding WT (white boxes), mtabcg56-1 (a), and mtabcg56-2 (b) (grey boxes), 21 days after inoculation (dai) with the E. meliloti 1021 strain. For each box-and-whiskers plot: the centre black line represents the median; a ‘+’ represents the mean; the box extends from the 25th to 75th percentiles; the whiskers are drawn down to the 10th percentile and up to the 90th. Points below and above the whiskers are drawn as individual dots. Identical or different letters indicate no or significant differences (P < 0.05), respectively; n represents the number of individual plants obtained from two independent experiments. P values, determined by the Kruskal-Wallis test with a post hoc Dunn’s multiple comparison test, can be found in Supplementary Data.

Source data

Extended Data Fig. 9 Relative expression of MtABCG56 homologues.

a, Relative expression of MtABCG37, 39, 57 in different M. truncatula organs from 6-week-old plants. The data represent mean ± s.d. of 6 biological replicates obtained from two independent experiments. b, Fold changes of MtABCG37, MtABCG39, MtABCG57, 72 hai with E. meliloti compared to the mock-treated control in WT-1 and mtabcg56-1 roots. The data represent mean ± s.d. of 3 biological replicates. Identical or different letters indicate no or significant differences (P < 0.05), respectively. P values, determined by two-way ANOVA with a post hoc Tamhane’s T2 multiple comparison test, can be found in Supplementary Data. c, The tissue-specific expression pattern of MtABCG37, MtABCG39 and MtABCG57, 72 hai with E.meliloti E65 strain, in WT-1 and mtabcg56-1 roots carrying the ProMtABCG37:GUS, ProMtABCG39:GUS, ProMtABCG57:GUS constructs, respectively. Images are representative of n > 10 roots obtained from two independent experiments (transformations). Bottom panels represent cross-section of GUS-stained roots. Scale bar, 100 µm. d, Relative expression of MtABCG37, 39, 57 in roots mock-treated or treated with 1 µM iP. The data represent mean ± s.d. of 6 biological replicates obtained from two independent experiments. Identical or different letters indicate no or significant differences (P < 0.05), respectively. P values, determined by Kruskal-Wallis test with a post hoc Dunn’s multiple comparison test, can be found in Supplementary Data.

Source data

Extended Data Fig. 10 Working model for the role of ABCG-driven CK transport in an early symbiotic interaction.

a, In wild-type M. truncatula, following NF perception, LOGs expression in the epidermis triggers cytokinin conversion from nucleotide precursors (CK-NT) to bioactive forms (CK)1. Concomitantly, NF-dependent up-regulation of MtENOD11 is a part of the symbiosis signalling pathway leading to initiation of infection events56,57. CK as a presumed non-cell autonomous signal1,2,6,7 activates CRE-dependent cortical responses. MtABCG56-driven transport participates in the efflux of CK from the epidermis. As a consequence, target gene transcription is initiated, including response regulators (RRs), MtABCG56, and de novo cytokinin biosynthesis takes place. The cortical cytokinins biosynthesized in response to microsymbiont inoculation trigger extension of nodulation responses within cortical cell layers, leading to nodule development20,29,30. The ABCG-driven CK export is a part of auto-activation mechanism. b, In mtabcg56 mutant, reduced ABCG-driven CK export from epidermis, negatively affects up-regulation of MtENOD11 and infection events. In root cortex, decreased/delayed CK response potentially results in reduced/delayed nodule organogenesis. Information, published elsewhere, concerning early nodulation stages are indicated by intact lines.

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Jarzyniak, K., Banasiak, J., Jamruszka, T. et al. Early stages of legume–rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins. Nat. Plants 7, 428–436 (2021). https://doi.org/10.1038/s41477-021-00873-6

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