How MtLICK1/2 Coordinate Symbiosis and Suppress Immunity in Medicago

Legumes depend on symbiotic interactions with rhizobia to meet their nitrogen needs. However, how these plants tolerate beneficial rhizobia while maintaining effective immunity against pathogens remains unclear.

On May 6, 2025, Prof. Ertao Wang’s group at the Chinese Academy of Sciences published a study in Nature titled “A kinase mediator of rhizobial symbiosis and immunity in Medicago”, revealing a novel mechanism underlying this balance. Using a Medicago yeast cDNA library constructed by Omics Empower, the team identified MtLICK1/2—a pair of cytoplasmic kinases that interact with the Nod factor receptor MtLYK3. These kinases coordinate both the establishment of symbiosis and the suppression of plant immune responses.

Key Methods

•  Yeast Library: Medicago truncatula cDNA expression library (constructed by Omics Empower)

•  Bait Protein: MtLYK3 (cytoplasmic domain)

•  Screening Method: Yeast two-hybrid (GAL4 system)

• Validation: Y2H assay, fluorescence co-localization, co-immunoprecipitation (Co-IP)

   *Service Provider：The Medicago yeast cDNA library used in this project was constructed by Omics Empower.If you're looking for similar yeast library solutions—including library        construction and screening—our team offers end-to-end support tailored to your research needs.

Major Findings

1. Identification of MtLICK1/2 as MtLYK3-Interacting Kinases

Through yeast two-hybrid screening, MtLICK1 was identified as a strong interactor of MtLYK3. Phylogenetic analysis revealed that MtLICK1 belongs to a conserved RLK subfamily found in arbuscular mycorrhizal plants. Its homolog MtLICK2 also interacts specifically with MtLYK3, forming a receptor complex at the plasma membrane.

Figure 1. Interaction between MtLYK3 and MtLICK1/2

2. MtLICK1/2 Are Essential for Rhizobial Symbiosis

To investigate whether MtLICK1 and MtLICK2 are involved in rhizobial symbiosis, the researchers first examined their expression levels. Both genes were significantly upregulated following Nod factor treatment or inoculation with Sinorhizobium meliloti. Promoter–GUS assays showed that pMtLICK1::GUS and pMtLICK2::GUS were primarily expressed in root hairs and the elongation zone. After rhizobial infection, their expression localized to nodule primordia and the infection zone of mature nodules, showing a similar pattern to pMtLYK3::GUS.

To further assess function, the team generated Mtlick1, Mtlick2, and Mtlick1/2 mutants. Upon inoculation with S. meliloti 1021, all mutants showed significantly reduced nodulation. In particular, Mtlick1/2 double mutants failed to form functional pink nodules, displaying only a few small bumps even after 21 or 28 days, with no detectable nitrogen fixation. Silencing MtLICK1 in the Mtlick2 background using RNAi led to a further decrease in nodulation, while genetic complementation restored the phenotype. Together, these results indicate that MtLICK1 and MtLICK2 function redundantly as key regulators of rhizobial infection and nodule development.

Figure 2. MtLICK1 and MtLICK2 are essential for rhizobial symbiosis

3. MtLICK1/2 Are Critical Signal Transducers in Nod Factor Pathways

To test whether MtLICK1/2 function as signaling kinases in the Nod factor pathway, the authors measured expression levels of symbiotic marker genes (MtMIN, MtENOD11). Compared to wild-type, the Mtlick1/2 double mutant showed significantly reduced expression, indicating a critical role for MtLICK1/2 in initiating symbiotic signaling.

In vitro kinase assays confirmed that both proteins exhibit autophosphorylation and transphosphorylation activity. Sequence alignment and site-directed mutagenesis identified key ATP-binding residues—K78 in MtLICK1 and K73 in MtLICK2—as essential for their kinase function (Fig. 3a).

4. MtLYK3 Phosphorylates MtLICK1/2 to Promote Symbiosis

Mass spectrometry identified three MtLYK3-mediated phosphorylation sites in MtLICK1/2, located within the P-loop and activation loop of their kinase domains. Phospho-mimic mutants (e.g., MtLICK1^S60D^, MtLICK2^S55D^, MtLICK1/2^3D^) exhibited enhanced kinase activity and fully rescued nodule formation in the Mtlick1/2 mutant. In contrast, non-phosphorylatable variants (e.g., MtLICK1/2^3A^) failed to restore function (Fig. 3b–e).

Further experiments showed that Nod factor perception activates MtLYK3, which then phosphorylates MtLICK1/2. Upon phosphorylation, MtLICK1/2 exhibit reduced interaction with MtLYK3—suggesting a release mechanism for downstream signaling (Fig. 3f–g).

Figure 3. MtLYK3 phosphorylates MtLICK1/2 to regulate rhizobial symbiosis.

5. MtLICK1/2 Phosphorylate MtLYK3 and Enhance Its Function

Given their transphosphorylation activity, the authors hypothesized that MtLICK1/2 might also activate MtLYK3. In vitro kinase assays confirmed this: catalytically inactive MtLYK3^K349E^ could not self-phosphorylate but was phosphorylated by recombinant MtLICK1^WT^ and MtLICK2^WT^ (Fig. 4a). MtLICK1/2 significantly enhanced MtLYK3 phosphorylation in both in vitro and in vivo assays (Fig. 4b).

Eight phosphorylation sites on MtLYK3 were identified, including T472, T475, T480, and Y483 within the activation segment. Alanine substitution at these sites impaired nodulation. Specifically, MtLYK3^Y483F^ lacked kinase activity and failed to rescue nodulation or gene expression in Mtlyk3-1 mutants (Fig. 4c–d). This mutant protein was also unstable, suggesting Y483 contributes to MtLYK3 stability.

Another key site, Y488, lies within the conserved YAQ motif, known to mediate symbiosis–immunity balance in Lotus. Anti-phospho-Y488 antibodies revealed that Nod factor-induced Y488 phosphorylation depends on MtLICK1/2 (Fig. 4e–f). The MtLYK3^Y488F^ mutant failed to activate symbiotic markers or restore nodulation (Fig. 4g–h), highlighting the essential role of Y488. Further, MtLYK3 phosphorylates the downstream kinase MtDMI2—but this was abolished in the Y488F mutant.

Non-phosphorylatable MtDMI2 variants also failed to restore nodulation, confirming the necessity of this phosphorylation cascade.

Figure 4. MtLICK1/2-mediated phosphorylation of MtLYK3 is essential for symbiosis.

6. MtLICK1/2 Suppress Immune Responses During Symbiosis

Compared to wild-type, Mtlick1, Mtlick2, and Mtlick1/2 mutants exhibited heightened immune responses after S. meliloti inoculation, including increased MAPK activation, ROS accumulation, and elevated expression of immune marker genes (Fig. 5a–d).

To assess broader immune function, plants were challenged with the pathogen Ralstonia solanacearum. Wild-type plants were more susceptible than the mutants, suggesting MtLICK1/2 normally suppress immunity to facilitate rhizobial colonization.

When wild-type plants were treated with both Nod factors and Ralstonia, susceptibility to infection increased. However, the Mtlick1/2 mutants showed no such enhancement, indicating that Nod factor–mediated immune suppression requires MtLICK1/2 (Fig. 5f).

Figure 5. MtLICK1/2 attenuate immune responses to promote symbiosis.

Summary

Figure 6. Dual roles of MtLICK1/2 in symbiotic signaling and immune regulation.

This study reveals that MtLICK1 and MtLICK2 interact specifically with the Nod factor receptor MtLYK3 and participate in reciprocal phosphorylation during the establishment of rhizobial symbiosis. Beyond initiating symbiotic signaling, MtLICK1/2 also fine-tune the plant immune response, ensuring compatibility with rhizobia while preventing overactivation of defense pathways. These findings provide new insight into how legumes coordinate symbiosis and immunity at the molecular level.

Reference

Wang, D., Jin, R., Shi, X. et al. A kinase mediator of rhizobial symbiosis and immunity in Medicago. Nature (2025). https://doi.org/10.1038/s41586-025-09057-0