Journal of Plant Research

, Volume 129, Issue 5, pp 909–919 | Cite as

Expression of the CLE-RS3 gene suppresses root nodulation in Lotus japonicus

  • Hanna Nishida
  • Yoshihiro Handa
  • Sachiko Tanaka
  • Takuya SuzakiEmail author
  • Masayoshi KawaguchiEmail author
Regular Paper


Cell-to-cell communication, principally mediated by short- or long-range mobile signals, is involved in many plant developmental processes. In root nodule symbiosis, a mutual relationship between leguminous plants and nitrogen-fixing rhizobia, the mechanism for the autoregulation of nodulation (AON) plays a key role in preventing the production of an excess number of nodules. AON is based on long-distance cell-to-cell communication between roots and shoots. In Lotus japonicus, two CLAVATA3/ESR-related (CLE) peptides, encoded by CLE-ROOT SIGNAL 1 (CLE-RS1) and -RS2, act as putative root-derived signals that transmit signals inhibiting further nodule development through interaction with a shoot-acting receptor-like kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1). Here, an in silico search and subsequent expression analyses enabled us to identify two new L. japonicus CLE genes that are potentially involved in nodulation, designated as CLE-RS3 and LjCLE40. Time-course expression patterns showed that CLE-RS1/2/3 and LjCLE40 expression is induced during nodulation with different activation patterns. Furthermore, constitutive expression of CLE-RS3 significantly suppressed nodule formation in a HAR1-dependent manner. TOO MUCH LOVE, a root-acting regulator of AON, is also required for the CLE-RS3 action. These results suggest that CLE-RS3 is a new component of AON in L. japonicus that may act as a potential root-derived signal through interaction with HAR1. Because CLE-RS2, CLE-RS3 and LjCLE40 are located in tandem in the genome and their expression is induced not only by rhizobial infection but also by nitrate, these genes may have duplicated from a common gene.


Autoregulation of nodulation CLE Legume Lotus japonicus Nodulation Root nodule symbiosis 



We thank Makoto Hayashi for providing M. loti MAFF303099 expressing DsRED. We also thank Satoru Okamoto for providing the p35S::CLE-RS1, p35S::CLE-RS2 and p35S::GUS plasmids. This work was supported by the National Institute for Basic Biology (NIBB) Core Research Facilities, the NIBB Model Plant Research Facility and by MEXT/JSPS KAKENHI, Japan (16H01457 to T.S. and 25291066 to M.K.).

Supplementary material

10265_2016_842_MOESM1_ESM.pdf (148 kb)
Supplementary tables (PDF 148 kb)
10265_2016_842_MOESM2_ESM.tif (148 kb)
Fig. S1 Schematic structure of the genomic region harboring CLE-RS2, CLE-RS3 and LjCLE40 (TIFF 148 kb)
10265_2016_842_MOESM3_ESM.tif (211 kb)
Fig. S2 Real-time RT-PCR analysis of CLE-RS3 (a), LjCLE40 (b), CLE-RS1 (c), CLE-RS2 (d), LjCLE39 (e), LjCLE41 (f) and LjCLE42 (g) expression in wild-type. Each cDNA sample was prepared from total RNA derived from the flower, leaf, stem, shoot apex, non-inoculated (-) and 1 dai (+) roots. The expression patterns of CLE-RS1 and CLE-RS2 in the organs other than inoculated roots are shown as inset (c, d). LjUBQ was used to assess the relative expression of each gene. Error bars indicate SE (n = 3 independent pools of respective organs) (TIFF 210 kb)
10265_2016_842_MOESM4_ESM.tif (172 kb)
Fig. S3 Real-time RT-PCR analysis of LjCLE39 (a), LjCLE41 (b) and LjCLE42 (c) expression in wild-type non-inoculated roots (0) and 1, 3, 5, 7 and 14 dai. Each cDNA was prepared from total RNA derived from the entire root. LjUBQ was used to assess the relative expression of each gene. Error bars indicate SE (n = 3–4 independent pools of roots) (TIFF 172 kb)
10265_2016_842_MOESM5_ESM.tif (80 kb)
Fig. S4 Real-time RT-PCR analysis of CLE-RS3 expression in stably transformed L. japonicus transgenic plants that were constitutively expressing CLE-RS3 or GUS. Each cDNA was prepared from total RNA derived from the entire root. LjUBQ was used to assess the relative expression of each gene. Error bars indicate SE (n = 3 independent pools of roots) (TIFF 80 kb)


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Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  1. 1.National Institute for Basic BiologyOkazakiJapan
  2. 2.School of Life ScienceThe Graduate University for Advanced StudiesOkazakiJapan
  3. 3.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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