Journal of Plant Research

, Volume 132, Issue 5, pp 641–653 | Cite as

ERN1 and CYCLOPS coordinately activate NIN signaling to promote infection thread formation in Lotus japonicus

  • Meng Liu
  • Takashi Soyano
  • Koji Yano
  • Makoto Hayashi
  • Masayoshi KawaguchiEmail author
Regular Paper


Legumes engage in symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia, under nitrogen-limited conditions. In many legumes, the root invasion of rhizobia is mediated by infection threads (ITs), tubular invaginations of the host cell wall and plasma membrane, developed from infection foci of deformed root hairs. IT formation is regulated by a series of signal transduction in host root. Nodulation signals activate the host transcription factor (TF), CYCLOPS, which directly induces expression of two TF genes, ERF REQUIRED FOR NODULATION1 (ERN1) and NODULE INCEPTION (NIN), essential for IT development. Here, we explored the relationship among these three symbiotic TF genes in the model legume Lotus japonicus and examined how their interplay contributes to IT formation. qRT-PCR analysis showed that NIN expression induced by rhizobial infection was attenuated in ern1-1, and further declined in cyclops-3 ern1-1. ERN1 overexpression led to induction of NIN expression in cyclops-3 ern1-1 in the presence of rhizobia. Thus, in addition to CYCLOPS, ERN1 is able to increase the NIN expression level depending on infection. Furthermore, consistent with this transcriptional hierarchy, ectopic expression of ERN1 as well as NIN suppressed the IT-deficient cyclops-3 phenotype, but ERN1 failed to confer ITs in the nin-2 root. However, the ern1-1 symbiotic epidermal phenotype was not suppressed by the NIN ectopic expression. The cyclops-3 ern1-1 double mutant was less sensitive to rhizobial infection than the single mutants and defective in the symbiotic root hair response at earlier stages. This more severe phenotype of the double mutant suggests a role for ERN1 that independent of the CYCLOPS-mediated transcriptional regulation. We conclude that ERN1 is involved in regulating NIN expression in addition to CYCLOPS, and these TFs coordinately promote the symbiotic root hair response and IT development. Our data help to reveal the extensive role of ERN1 in root nodule symbiosis signaling.


CYCLOPS ERN1 Lotus japonicus NIN Root nodule symbiosis 





Calcium and calmodulin-dependent protein kinase












Nod factor receptor




Quantitative real-time PCR


Transcription factor


Wild type



We thank Dr. Emiko Yoro in Rikkyo Univeisity for her advice on the manuscript. And we thank A.T., the National Institute for Basic Biology’s Functional Genomics Facility and Model Plant Research Facility for technical supports. This work was supported by Ministry of Education, Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research [Grant numbers 22128006, 17H03702 to M.K.]; and Sumitomo Foundation Grant for Basic Science Research [150574 to T.S.]. This work was also supported in part by SOKENDAI (The Graduate University for Advanced Studies).

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Supplementary material

10265_2019_1122_MOESM1_ESM.pdf (583 kb)
Supplementary material 1 (PDF 582 kb)


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Copyright information

© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • Meng Liu
    • 1
    • 2
  • Takashi Soyano
    • 1
    • 2
  • Koji Yano
    • 1
  • Makoto Hayashi
    • 3
  • Masayoshi Kawaguchi
    • 1
    • 2
    Email author
  1. 1.Division of Symbiotic SystemsNational Institute for Basic BiologyOkazakiJapan
  2. 2.Department of Basic Biology, School of Life ScienceSOKENDAI (The Graduate University for Advanced Studies)OkazakiJapan
  3. 3.Center for Sustainable Resource Science, RIKENYokohamaJapan

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