id: Q8L4H4 gene_symbol: NORK product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:3880 label: Medicago truncatula description: >- NORK (Nodulation receptor kinase; also DMI2, DOES NOT MAKE INFECTIONS 2, and MtSYMRK) is a single-pass transmembrane receptor-like serine/threonine protein kinase of Medicago truncatula with a signal peptide, an extracellular malectin-like domain followed by leucine-rich repeats, a transmembrane helix and a cytoplasmic kinase domain. It is a core component of the common symbiosis signalling pathway (CSSP) shared by the rhizobial root-nodule symbiosis and the arbuscular mycorrhizal (AM) symbiosis: dmi2 null mutants are blocked in Nod factor-induced root hair responses, nodulin gene expression, cortical cell division and nuclear-associated calcium spiking, and fail to form both nodules and AM associations. NORK acts downstream of the LysM receptor kinases (NFP/LYK3 for Nod factors) and upstream of DMI1-dependent calcium spiking and the nuclear decoder CCaMK/DMI3. No ligand for its ectodomain has been identified. The recombinant kinase domain autophosphorylates and trans-phosphorylates substrates in vitro, including the E3 ubiquitin ligase PUB1 (a negative regulator of both symbioses) and the LysM receptor kinase LYK3; it also binds HMG-CoA reductase 1. NORK localises to the plasma membrane of root cells and to the membrane surrounding infection threads in nodules, and is additionally required in nodules for release of rhizobia from infection threads and symbiosome formation. The two symbiotic roles are genetically separable: the kinase-inactive G795E (R38) allele abolishes nodulation but leaves mycorrhization intact. alternative_records: - id: A9YWR7 relationship: REFERENCE_PROTEOME_EQUIVALENT reference_proteome: UP000002051 reviewed_on: '2026-10-19' reason: Retain Q8L4H4 as the primary Swiss-Prot accession. The reference sequence is exactly identical across all 925 residues. G7KGF4 is an additional shorter model with 902 identical residues, but A9YWR7 represents the complete primary sequence. The kinase-family search reached its 1000-hit cap; this does not weaken the observed full-length identity but prevents claiming exhaustive paralog retrieval. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 57,093 sequences across UP000002051 at E <= 1e-5 and sensitivity 7.5, returning 1000 hits. Every returned sequence was globally aligned using BLOSUM62 with gap-open -10 and extension -0.5. Scores and sequences for all 1000 returned candidates are retained, with 10 detailed alignment files. The heuristic search does not establish genomic absence or unique locus identity. The search reached its 1000-hit retrieval cap. supported_by: - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: 'target_sequence_count: 57093' - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: 'candidate_count: 1000' - type: SEQUENCE_COMPARISON description: 'A9YWR7: 925 identities across 925 global-alignment columns (100.0% identity including gaps), 0 substitutions and 0 gap columns. Query coverage is 100.0%; target coverage is 100.0%. Sequence similarity is interpreted in the manual rationale. Identity over paired residues is 100.0000% (925/925); gap columns are excluded from this denominator.' supported_by: - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: |- query_length: 925 target_length: 925 alignment_columns: 925 identities: 925 mismatches: 0 gap_columns: 0 identity_percent: 100.0 query_coverage_percent: 100.0 target_coverage_percent: 100.0 - type: PROTEOME_MEMBERSHIP description: The primary accession is outside every searched exact-taxon reference proteome. Current UniProt records place A9YWR7 in UP000002051 and give it the same taxon as the primary. supported_by: - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: 'alternative_in_reference_proteome: true' - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: 'same_taxon: true' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 11-NOV-2015, entry version 79.: no Proteomes cross-reference; 07-OCT-2020, entry version 105.: no Proteomes cross-reference; 27-NOV-2024, entry version 119.: no Proteomes cross-reference. These dated observations do not establish uninterrupted membership/absence, the exact change date, or UniProt''s motivation.' supported_by: - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: |- entry_date: 11-NOV-2015, entry version 79. proteomes: [] - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: |- entry_date: 07-OCT-2020, entry version 105. proteomes: [] - reference_id: file:MEDTR/NORK/NORK-record-evidence.yaml supporting_text: |- entry_date: 27-NOV-2024, entry version 119. proteomes: [] existing_annotations: - term: id: GO:0004672 label: protein kinase activity evidence_type: IDA original_reference_id: PMID:26839127 qualifier: enables review: summary: >- Recombinant DMI2/NORK kinase domain autophosphorylates and directly phosphorylates the E3 ubiquitin ligase PUB1 in vitro (Vernie et al. 2016); independently confirmed by Jayaraman et al. 2017, where NORK trans-phosphorylates casein and kinase-dead LYK3. action: ACCEPT reason: >- Direct biochemical evidence of protein kinase activity on the target itself. The term is general (the more specific serine/threonine kinase terms are also annotated) but correct, and the curator-asserted IDA is consistent with all available data. supported_by: - reference_id: PMID:26839127 supporting_text: >- we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway - reference_id: PMID:28267253 supporting_text: >- the detection of the LYK3 band implies that LYK3 is indeed trans-phosphorylated by NORK - term: id: GO:0004672 label: protein kinase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR001245 - InterPro:IPR008271 review: summary: >- InterPro2GO mapping from the Ser/Thr kinase catalytic domain and active site signatures. NORK has a canonical kinase domain (residues 596-873) with a predicted catalytic aspartate and ATP-binding lysine. action: ACCEPT reason: >- Electronic inference fully consistent with experimental kinase activity on NORK itself (PMID:26839127, PMID:28267253). supported_by: - reference_id: PMID:28267253 supporting_text: >- As expected, the wild-type versions of LYK3 and NORK exhibited auto-phosphorylation (Figure 3A and 3B) and trans-phosphorylation (as indicated by the phosphorylation of casein) activities - term: id: GO:0004674 label: protein serine/threonine kinase activity evidence_type: IEA original_reference_id: GO_REF:0000003 qualifier: enables supporting_entities: - EC:2.7.11.1 review: summary: >- EC 2.7.11.1 mapping. UniProt records both serine and threonine phosphorylation (RHEA:17989 and RHEA:46608) from Vernie et al. 2016, and Jayaraman et al. 2017 mapped NORK-phosphorylated serines on LYK3 and NORK autophosphorylation sites including Ser and Thr residues. action: ACCEPT reason: >- Correct and appropriately specific description of the catalytic activity; this is the core molecular function of NORK. supported_by: - reference_id: PMID:28267253 supporting_text: >- We identified that sites Ser-269, Ser-273, Ser-307, Ser-323 and Ser-471 of LYK3 (as a control only mLYK3 was used) were phosphorylated by NORK using Mass spectrometry - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:26839127 qualifier: enables supporting_entities: - UniProtKB:E4NKF8 review: summary: >- Interaction with PUB1 (E4NKF8), a U-box E3 ubiquitin ligase, shown by yeast two-hybrid and co-immunopurification; PUB1 is also a phosphorylation substrate of NORK. action: MODIFY reason: >- The interaction is real but "protein binding" is uninformative. The cited paper establishes that the partner is an E3 ubiquitin ligase, so the more informative "ubiquitin protein ligase binding" (GO:0031625) is directly supported by the same evidence. The functional relationship is kinase-substrate (NORK phosphorylates PUB1); PUB1 did not detectably ubiquitinate DMI2 according to the deep-research summary of the full text. proposed_replacement_terms: - id: GO:0031625 label: ubiquitin protein ligase binding supported_by: - reference_id: PMID:26839127 supporting_text: >- PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase - reference_id: PMID:26839127 supporting_text: >- we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2 - reference_id: file:MEDTR/NORK/NORK-deep-research-falcon.md supporting_text: >- Yeast two-hybrid and co-immunopurification support DMI2–PUB1 binding; recombinant DMI2 autophosphorylates and directly phosphorylates PUB1 in vitro - term: id: GO:0005524 label: ATP binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR000719 - InterPro:IPR017441 review: summary: >- Kinase domain ATP-binding signature (residues 602-610 and Lys624). ATP binding is intrinsic to the demonstrated kinase activity. action: ACCEPT reason: >- Correct structural inference for an active protein kinase; non-informative beyond the kinase activity terms but not wrong. supported_by: - reference_id: PMID:28267253 supporting_text: >- As expected, the wild-type versions of LYK3 and NORK exhibited auto-phosphorylation - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0162 review: summary: >- UniProt subcellular-location mapping (single-pass membrane protein, by similarity/topology). NORK has a signal peptide and a single transmembrane helix (522-542). action: ACCEPT reason: >- Correct but general. Experimental localisation shows NORK is at the plasma membrane and the infection-thread membrane (PMID:16006515); a NEW plasma membrane annotation is proposed below to capture the specific location. supported_by: - reference_id: PMID:16006515 supporting_text: >- The protein locates to the host cell plasma membrane and to the membrane surrounding the infection threads. - term: id: GO:0046777 label: protein autophosphorylation evidence_type: IDA original_reference_id: PMID:26839127 qualifier: involved_in review: summary: >- Purified DMI2 kinase domain autophosphorylates in vitro (Vernie et al. 2016); autophosphorylation was also shown by Jayaraman et al. 2017, who mapped at least 37 NORK phosphosites and showed the G794E (R38) mutant lacks autophosphorylation. action: ACCEPT reason: >- Directly demonstrated on the target. Autophosphorylation is a regulatory property of the kinase (SYMRK kinase activity depends on its phosphorylation status) rather than its biological output, but the annotation is accurate. supported_by: - reference_id: file:MEDTR/NORK/NORK-deep-research-falcon.md supporting_text: >- Purified NORK/DMI2 intracellular kinase autophosphorylates and trans-phosphorylates substrates; kinase-dead G794E lacks activity - reference_id: PMID:28267253 supporting_text: >- At least 37 phosphorylation sites of NORK were identified in this study - term: id: GO:0106310 label: protein serine kinase activity evidence_type: EXP original_reference_id: PMID:26839127 qualifier: enables review: summary: >- UniProt Rhea-based curation (RHEA:17989) from the PUB1 trans-phosphorylation and autophosphorylation assays of Vernie et al. 2016. Serine phosphorylation of a substrate by NORK is independently confirmed by MS-mapped serines on LYK3 (Jayaraman et al. 2017). action: ACCEPT reason: >- Supported by direct biochemical data; consistent with the serine/threonine kinase classification. supported_by: - reference_id: PMID:26839127 supporting_text: >- PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2 - reference_id: PMID:28267253 supporting_text: >- We identified that sites Ser-269, Ser-273, Ser-307, Ser-323 and Ser-471 of LYK3 (as a control only mLYK3 was used) were phosphorylated by NORK - term: id: GO:0106310 label: protein serine kinase activity evidence_type: IEA original_reference_id: GO_REF:0000116 qualifier: enables supporting_entities: - RHEA:17989 review: summary: >- Electronic mapping from the UniProt Rhea catalytic-activity annotation, itself experimentally based (PMID:26839127). action: ACCEPT reason: Consistent with experimental evidence for serine phosphorylation by NORK. supported_by: - reference_id: PMID:28267253 supporting_text: >- We identified that sites Ser-269, Ser-273, Ser-307, Ser-323 and Ser-471 of LYK3 (as a control only mLYK3 was used) were phosphorylated by NORK # --- Proposed NEW annotations --- - term: id: GO:0009877 label: nodulation evidence_type: IMP original_reference_id: PMID:11006338 review: summary: >- dmi2 mutants are Nod- and blocked in all early Nod factor responses (root hair deformation, nodulin expression, cortical cell division) and in Nod factor-induced calcium spiking; reduced DMI2 expression in nodules blocks release of rhizobia from infection threads and symbiosome formation. NORK is the receptor-kinase step of the Nod factor signal transduction pathway. action: NEW reason: >- Nodulation is the best-established biological role of NORK and is absent from GOA. Participation test - NORK is not a substrate or passive requirement; it is an active signal-transducing kinase in the Nod factor signalling cascade that phosphorylates pathway components (LYK3, PUB1) and whose kinase activity is specifically required for nodulation (the kinase-dead G795E/R38 allele is Nod- but Myc+). Comparator check - the other Medicago signalling components in the same pathway carry this term: LYK3 (Q6UD73, IMP PMID:12947035), CCaMK/DMI3 (Q6RET7, IMP PMID:22874912) and NFP. Its absence from NORK is a curation gap rather than a convention. supported_by: - reference_id: PMID:11006338 supporting_text: >- Mutants in four genes ( DMI1 , DMI2 , DMI3 , and NSP ) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor–activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. - reference_id: PMID:11078514 supporting_text: >- We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. - reference_id: PMID:16006515 supporting_text: >- At least one of these genes, encoding the receptor kinase DOES NOT MAKE INFECTIONS 2 (DMI2) is essential for symbiosome formation. - reference_id: PMID:28267253 supporting_text: >- had no effect on mycorrhization of plants but impaired the ability of plants to form nodules - term: id: GO:0036377 label: arbuscular mycorrhizal association evidence_type: IMP original_reference_id: PMID:11006338 review: summary: >- dmi2 mutants of M. truncatula cannot establish AM symbiosis; NORK/SYMRK is the receptor-kinase entry point of the common symbiosis signalling pathway used by both rhizobia and AM fungi. action: NEW reason: >- The AM role is equally well established genetically and is missing from GOA. Kept as a distinct process term from nodulation because the two are genetically separable (G795E/R38 abolishes nodulation without affecting mycorrhization), so NORK's AM function does not rely on the same kinase requirement. Participation - NORK is a signalling component of the CSSP that transduces the symbiotic signal towards nuclear calcium spiking. Comparator - rice CCaMK, a downstream CSSP component, carries this term in this repository's review (genes/ORYSJ/CCAMK). Direct binding of a Myc factor to NORK is not established, so no receptor/ligand-binding term is proposed. supported_by: - reference_id: PMID:11006338 supporting_text: >- dmi1 , dmi2 , and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula - reference_id: PMID:28267253 supporting_text: >- Also, NORK is essential for the formation of a symbiotic association with arbuscular mycorrhizal fungi - reference_id: PMID:26839127 supporting_text: >- the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses - term: id: GO:0005886 label: plasma membrane evidence_type: IDA original_reference_id: PMID:16006515 review: summary: >- Immunolocalisation/fusion analysis in M. truncatula nodules places DMI2 at the host cell plasma membrane and the infection-thread membrane. action: NEW reason: >- More specific, experimentally supported location than the existing IEA "membrane" annotation; consistent with a single-pass receptor-like kinase. The comparator LYK3 carries plasma membrane by IDA. supported_by: - reference_id: PMID:16006515 supporting_text: >- The protein locates to the host cell plasma membrane and to the membrane surrounding the infection threads. core_functions: - description: >- Membrane-anchored receptor-like serine/threonine protein kinase whose cytoplasmic kinase domain autophosphorylates and trans-phosphorylates symbiotic signalling partners, including the E3 ubiquitin ligase PUB1 and the LysM receptor kinase LYK3. No extracellular ligand has been identified. molecular_function: id: GO:0004674 label: protein serine/threonine kinase activity locations: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: PMID:28267253 supporting_text: >- the detection of the LYK3 band implies that LYK3 is indeed trans-phosphorylated by NORK - reference_id: PMID:26839127 supporting_text: >- PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2 - reference_id: PMID:16006515 supporting_text: >- The protein locates to the host cell plasma membrane and to the membrane surrounding the infection threads. - description: >- Nodulation: the kinase-dependent receptor step of Nod factor signal transduction, acting downstream of NFP/LYK3 and upstream of nuclear calcium spiking (DMI1) and CCaMK/DMI3; required for root hair responses, nodulin induction, cortical cell division, infection, and later for release of rhizobia from infection threads and symbiosome formation in nodules. Kinase activity is specifically required (G795E/R38 is Nod- Myc+). molecular_function: id: GO:0004674 label: protein serine/threonine kinase activity directly_involved_in: - id: GO:0009877 label: nodulation locations: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: PMID:11078514 supporting_text: >- We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. - reference_id: PMID:16006515 supporting_text: >- At least one of these genes, encoding the receptor kinase DOES NOT MAKE INFECTIONS 2 (DMI2) is essential for symbiosome formation. - reference_id: PMID:28267253 supporting_text: >- Here we demonstrate that the kinase activity is indeed abolished in the R38 mutant’s version of NORK - description: >- Arbuscular mycorrhizal association: common symbiosis signalling pathway component required for AM fungal colonisation, upstream of nuclear calcium spiking. This role is genetically distinct from nodulation, since a kinase-inactive allele that abolishes nodulation leaves mycorrhization intact. directly_involved_in: - id: GO:0036377 label: arbuscular mycorrhizal association locations: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: PMID:11006338 supporting_text: >- dmi1 , dmi2 , and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi - reference_id: PMID:28267253 supporting_text: >- had no effect on mycorrhization of plants but impaired the ability of plants to form nodules proposed_new_terms: [] suggested_questions: - question: >- What is the ligand (if any) of the NORK malectin-like/LRR ectodomain, and is ectodomain cleavage/release of the malectin-like domain a regulated step in rhizobial and AM signalling? - question: >- Why is NORK kinase activity required for nodulation but apparently dispensable for AM colonisation (G795E/R38 is Nod- Myc+)? Does NORK act as a scaffold for AM signalling, or does residual activity suffice? - question: >- Is the in vitro phosphorylation of LYK3 and PUB1 by NORK relevant in vivo, and what links NORK at the plasma membrane to DMI1-dependent nuclear calcium spiking (e.g. HMGR1-derived mevalonate)? suggested_experiments: - description: >- Complement M. truncatula dmi2 null mutants with kinase-dead and phosphosite-mutant NORK variants and score nodulation, AM colonisation and nuclear calcium spiking separately. hypothesis: >- NORK kinase activity is required for the nodulation branch but not for AM signalling, so the two symbiotic outputs depend on different NORK activities. experiment_type: genetic complementation with structure-function mutants - description: >- Phosphoproteomics of roots expressing wild-type versus kinase-dead NORK after Nod factor or Myc-LCO treatment to identify in vivo substrates. hypothesis: >- LYK3 and PUB1 are phosphorylated in a NORK-dependent manner in planta. experiment_type: quantitative phosphoproteomics references: - id: file:MEDTR/NORK/NORK-record-evidence.yaml title: Compact archived record and sequence evidence for Q8L4H4 - id: file:MEDTR/NORK/NORK-deep-research-falcon.md title: Falcon (Edison Scientific) deep research report for NORK findings: [] - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000003 title: Gene Ontology annotation based on Enzyme Commission mapping findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000116 title: Automatic Gene Ontology annotation based on Rhea mapping findings: [] - id: PMID:26839127 title: PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Abstract-only in cache. Source of the IDA/EXP kinase, autophosphorylation and PUB1 IPI annotations; abstract confirms DMI2 interacts with and phosphorylates PUB1. - id: PMID:12087406 title: A receptor kinase gene regulating symbiotic nodule development. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Cloning of NORK (M. sativa, with M. truncatula alleles); source of UniProt mutagenesis data including the Nod- Myc+ G795E allele. - id: PMID:12087405 title: A plant receptor-like kinase required for both bacterial and fungal symbiosis. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Lotus japonicus and pea SYMRK orthologs; establishes the dual bacterial/fungal requirement for the orthologous gene, not M. truncatula. - id: PMID:18156218 title: 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: HMGR1 is an interactor of the active NORK kinase domain. - id: PMID:28267253 title: Identification of the phosphorylation targets of symbiotic receptor-like kinases using a high-throughput multiplexed assay for kinase specificity. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text. NORK autophosphorylation, casein and LYK3 trans-phosphorylation; G794E (R38) kinase-dead and Nod- Myc+. - id: PMID:16006515 title: Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Plasma membrane/infection thread membrane localisation; symbiosome formation. - id: PMID:11078514 title: Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: DMI2 required for Nod factor-induced calcium spiking. - id: PMID:16134899 title: Expression of the Medicago truncatula DM12 gene suggests roles of the symbiotic nodulation receptor kinase in nodules and during early nodule development. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: DMI2 expression pattern in roots and nodule primordia. - id: PMID:11006338 title: Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: dmi2 mutants are Nod- and Myc-; defines DMI2 in the Nod factor transduction pathway.