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Autophosphorylation at the C-terminus of the DMI3 calmodulin-binding domain dismantles CaM-DMI3-IPD3 to initiate root

Yiting Zhou1, Yuchen Qian1, Yiang Wang1

  • 1College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China.

Plant Physiology
|June 9, 2026
PubMed

Insights

DMI3 protein phosphorylation controls root nodule symbiosis. A specific triplet motif acts as a switch, requiring hyperphosphorylation to activate symbiotic signaling and promote plant-microbe interactions.

Area of Science:

  • Plant molecular biology
  • Microbiology
  • Biochemistry

Background:

  • DMI3 (Doesn't Make Infections 3) is a calcium/calmodulin-dependent protein kinase (CCaMK) crucial for initiating root nodule symbiosis (RNS).
  • DMI3 decodes rhizobia-induced calcium signals by phosphorylating its partner, IPD3.
  • The exact mechanism of DMI3 activation in RNS remains to be fully elucidated.

Purpose of the Study:

  • To investigate the precise mechanism by which DMI3 activates the RNS pathway.
  • To identify the role of phosphorylation at a C-terminal triplet motif in DMI3 function.
  • To clarify the dual role of DMI3 as a kinase and scaffold in RNS.

Main Methods:

  • Site-directed mutagenesis to create phosphomimic and non-phosphorylated mutants of the DMI3 triplet motif (S343S344T345).
  • Analysis of DMI3 autophosphorylation (auto-P) and interaction with Ca2+/CaM and IPD3.
  • Assays for IPD3-triggered MtNIN transcription and nodulation.

Main Results:

  • Phosphorylation of the triplet motif acts as a molecular switch, requiring simultaneous phosphorylation of at least two residues for symbiotic activity.
  • Phosphomimic mutations enhanced DMI3 auto-P but impaired Ca2+/CaM and IPD3 binding.
  • DMI3 interaction with IPD3 suppressed MtNIN transcription, indicating a negative regulatory role by sequestering activated IPD3.
  • A model is proposed where repeated auto-P at the triplet dissociates the CaM-DMI3-IPD3 complex, releasing activated IPD3.

Conclusions:

  • The DMI3 triplet motif functions as a regulatory switch, transitioning from a non-phosphorylated to a hyperphosphorylated state to activate RNS.
  • DMI3 acts dually as a kinase and a scaffold, with autophosphorylation at the triplet motif licensing IPD3 activation.
  • This mechanism clarifies how DMI3 initiates the downstream transcriptional cascade essential for root nodule symbiosis.

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