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

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Abstract
The calcium/calmodulin-dependent protein kinase (CCaMK) DMI3 (Doesn't Make Infections 3) decodes rhizobia-induced Ca2+ signals through phosphorylation of its interacting partner, IPD3, thereby initiating root nodule symbiosis (RNS). However, the precise mechanism by which DMI3 activates this pathway remains unclear. Here, we show that phosphorylation of a triplet motif (S343S344T345) located at the C-terminus of the calmodulin-binding domain acts as a molecular switch. We identified T345 as a phosphorylation site that exhibits regulatory functions similar to those of S343 and S344. Phosphomimic mutations at any single residue of the 3 sites blocked DMI3-dependent nodulation, indicating that the kinase must initially remain nonphosphorylated at these positions. Conversely, simultaneous phosphorylation of at least 2 residues was required for symbiotic activity, revealing a transition from a phosphorylation-free state to a hyperphosphorylated status (simultaneous phosphorylation of at least 2 residues) at these sites. Phosphomimic mutations within the triplet enhanced DMI3 autophosphorylation (auto-P) but severely impaired its interaction with Ca2+/CaM and IPD3. In addition, the physical interaction between DMI3 and IPD3 suppressed IPD3-triggered MtNIN transcription and nodulation, indicating that DMI3 also acts as a negative regulator by sequestering activated IPD3. Thus, we propose a model in which repeated auto-P at the triplet contributes to RNS activation by dissociating the CaM-DMI3-IPD3 complex, thereby releasing phosphorylated and activated IPD3 to initiate the downstream transcriptional cascade. Our findings uncover a dual role for DMI3—as both a kinase and a scaffold—and clarify how auto-P within the triplet motif licenses IPD3 to trigger RNS.
Funding Information
  • National Natural Science Foundation of China (31970286)
  • National Natural Science Foundation of China (U1130304)
  • Natural Science Foundation of Zhejiang Province (LY22C020001)
  • Natural Science Foundation of Zhejiang Province (LY17C020004)
  • Natural Science Foundation of Zhejiang Province (LR20C020001)
  • Zhejiang Provincial Natural Science Foundation of China (LHZSZ24C010001)
  • Zhejiang provincial science and Technology Department (2025SDXT004-2)
  • Zhejiang Provincial Natural Science Foundation of China (LHZQN25C130006)
  • Hangzhou Science and Technology Bureau (20170432B01)
  • Hangzhou Science and Technology Bureau (20180432B11)
  • State Key Laboratory of Subtropical Silviculture (SKLSS-KF2024-08)

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