OsCCaMK / OsDMI3 (Q6AVM3; Os05g0489900, LOC_Os05g41090) is the rice ortholog of the legume common-symbiosis-pathway kinase DMI3/CCaMK, a calcium- and calcium/calmodulin-dependent serine/threonine protein kinase (EC 2.7.11.17). It has an N-terminal protein-kinase domain, a calmodulin-binding/autoinhibitory region, and three C-terminal EF-hand Ca2+-binding motifs that let it act as an intracellular decoder of nuclear Ca2+ spiking in the Common Symbiosis Signaling Pathway. In rice, the demonstrated symbiotic role of OsCCaMK is in arbuscular mycorrhizal symbiosis; rice is a non-nodulating cereal and does not form nitrogen-fixing root nodules with rhizobia. OsCCaMK can restore both mycorrhization and rhizobial nodulation when expressed heterologously in legume dmi3/ccamk mutants, but rice itself has no nodulation program for the kinase to act in. OsDMI3 also has a separable abiotic-stress role in ABA signaling upstream of OsMPK1 and OsRBOHB during water-deprivation stress, and localizes to the nucleus, cytoplasm, and plasma membrane.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009877 nodulation | IEA GO_REF:0000043 | MODIFY | Summary: SPKW (GO_REF:0000043) annotation derived from the UniProt keyword "Nodulation"; snapshot-only, removed in the current GOA release. "Nodulation" (formation of nitrogen-fixing root nodules in symbiosis with rhizobia) is a process that rice - a cereal in the Poaceae - does NOT perform. The keyword is inherited from CCaMK/DMI3 orthologs in legumes, where the same kinase decodes Ca2+ spiking for BOTH nodulation and mycorrhization. In rice the kinase's genuine symbiotic role is arbuscular mycorrhizal (AM) symbiosis, not nodulation. Reason: Removal of the bare "nodulation" term is JUSTIFIED for rice, but the underlying symbiotic biology should be retained, not lost - so the correct curation is to MODIFY to a process rice actually performs. Rice is non-nodulating; the kinase decodes nuclear Ca2+ spiking in the common symbiosis pathway and, in rice, is genetically required for ARBUSCULAR MYCORRHIZAL symbiosis (Osccamk mutants are Myc-defective) [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. The "Nodulation" keyword reflects the conserved CCaMK family signature in legumes (rice OsCCaMK can even restore nodulation when transferred into a legume dmi3 mutant), but applying "nodulation" to the rice gene is a pathway/organism-context error directly analogous to the PPC16 photosynthesis- keyword case in this subproject: a family-signature keyword applied to a species that does not carry out the process. The annotation should be MODIFIED to "arbuscular mycorrhizal association" (GO:0036377), which the rice gene genuinely supports. Proposed replacements: arbuscular mycorrhizal association Supporting Evidence: PMID:17965173 We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md Loss of rice CCAMK strongly impairs **arbuscular mycorrhizal colonization**, with fungal entry defects and failure of proper cortical colonization |
| GO:0035556 intracellular signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation propagated across the CaMK Ser/Thr kinase phylogenetic group. CCaMK is an intracellular Ca2+/CaM-dependent kinase that transduces calcium signals to downstream effectors (CYCLOPS in symbiosis; OsMPK1/OsRBOHB in ABA-ROS signaling). Reason: Correct and at an appropriate (if general) level of specificity. OsCCaMK is the intracellular decoder of nuclear Ca2+ spiking, converting a calcium signature into downstream transcriptional/enzymatic outputs [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. It also transduces ABA/H2O2 signals via OsMPK1 [PMID:22869603]. "Intracellular signal transduction" is a generic but accurate parent term; more specific symbiotic-signaling processes are captured by the AM-association term (GO:0036377) below. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md decodes intracellular/nuclear calcium signatures and couples them to transcriptional programs and stress signaling outputs |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation from InterPro (IPR000719 protein kinase domain; IPR008271 Ser/Thr kinase active site). OsCCaMK has a bona fide N-terminal protein-kinase domain and is an active Ser/Thr kinase. Reason: Correct but a broad parent term. OsCCaMK/OsDMI3 is an active serine/threonine protein kinase (EC 2.7.11.17) whose kinase domain (residues 13-298) and ATP-binding/active-site residues are annotated in UniProt, and rice OsDMI3 kinase activity has been assayed directly [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. The more informative, specific MF (calcium/calmodulin-dependent protein kinase activity, GO:0004683) is also annotated; the generic parent is not wrong and can be accepted. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md OsDMI3 was immunoprecipitated and assayed using **myelin basic protein (MBP)** as an in vitro substrate, confirming enzymatic activity consistent with a Ser/Thr protein kinase |
| GO:0004683 calcium/calmodulin-dependent protein kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: IEA annotation from EC mapping (EC 2.7.11.17). This is the core, specific molecular function of OsCCaMK/OsDMI3: a Ca2+/calmodulin-dependent serine/threonine protein kinase. Reason: This is the accurate, specific molecular function and is also independently supported by IBA (GO_Central) and by the protein name itself ("Calcium and calcium/calmodulin- dependent serine/threonine-protein kinase"). Rice OsDMI3 kinase activity was measured by in-gel kinase assay using MBP as substrate in the presence of Ca2+ and calmodulin [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. This is a core function annotation. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md OsDMI3 was immunoprecipitated and assayed using **myelin basic protein (MBP)** as an in vitro substrate, confirming enzymatic activity consistent with a Ser/Thr protein kinase file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md The kinase reaction mixture included **0.5 mM CaCl2** and **2 mM calmodulin** |
| GO:0005509 calcium ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation from InterPro (IPR002048 EF-hand domain). OsCCaMK has three C-terminal EF-hand Ca2+-binding motifs that bind calcium and underlie its Ca2+-sensing function. Reason: Correct and core. UniProt annotates three EF-hand domains (residues ~392-505) with numerous Ca2+-coordinating BINDING residues, and CCaMK/DMI3 proteins are described as having C-terminal EF-hand Ca2+-binding motifs that enable regulation by Ca2+ signatures [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. Calcium binding via the EF-hands is mechanistically central to the kinase's role as a Ca2+ decoder. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md an N-terminal **kinase domain**, a **CaM-binding/autoinhibitory region**, and a C-terminal **EF-hand Ca2+-binding region** |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation from InterPro (IPR000719 protein kinase domain; IPR017441 protein kinase ATP-binding site). As a protein kinase, OsCCaMK binds ATP as the phosphate donor. Reason: Correct. UniProt annotates an ATP-binding region (residues 19-27) and an ATP-binding residue (43) in the kinase domain, consistent with the kinase mechanism (transfer of phosphate from ATP to protein Ser/Thr residues) [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. ATP binding is an obligatory cofactor-binding function for the kinase activity. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-uniprot.txt BINDING 19..27 FT /ligand="ATP" |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation (UniProtKB Subcellular Location keyword mapping, SL-0191). Nuclear localization is directly confirmed in rice by experimental subcellular-localization analysis and is mechanistically essential for the symbiotic Ca2+-spiking decode. Reason: Strongly supported and a core localization. OsDMI3 was experimentally localized to the nucleus (alongside cytoplasm and plasma membrane) [PMID:22869603], and nuclear localization of active CCaMK is required to activate symbiotic responses (the nuclear Ca2+ spiking it decodes is nuclear/perinuclear) [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. Duplicates the EXP nucleus annotation. Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md CCaMK is consistently framed as a **nuclear calcium-spiking decoder** in CSSP models, acting with the transcription factor CYCLOPS |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation (UniProtKB Subcellular Location keyword mapping, SL-0086). Cytoplasmic localization is confirmed in rice experimentally. Reason: Supported by direct rice evidence: OsDMI3 was localized to the nucleus, the cytoplasm and the plasma membrane [PMID:22869603]. Duplicates the EXP cytoplasm annotation. The cytoplasmic pool is consistent with its role in ABA/ROS stress signaling in addition to the nuclear symbiotic-signaling role. Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation (UniProtKB Subcellular Location keyword mapping, SL-0039; peripheral membrane protein by ECO:0000305). Plasma-membrane association is reported in rice. Reason: Supported by direct rice evidence: OsDMI3 was localized to the nucleus, the cytoplasm and the plasma membrane [PMID:22869603]; UniProt classifies it as a peripheral membrane protein at the cell membrane. Duplicates the EXP plasma membrane annotation. The peripheral plasma-membrane pool may relate to proximity to ABA/ROS signaling components (e.g. the membrane NADPH oxidase OsRBOHB). Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: IEA annotation from RHEA (RHEA:17989) via the EC 2.7.11.17 catalytic activity. Captures the serine-directed component of the kinase's protein-phosphorylation activity. Reason: Correct. The UniProt CATALYTIC ACTIVITY block records phosphorylation of both L-seryl-[protein] (RHEA:17989) and L-threonyl-[protein] (RHEA:46608) under EC 2.7.11.17, and OsDMI3 is an active Ser/Thr kinase assayed on MBP substrate [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. This Rhea-derived MF is consistent with the more general protein-kinase and the specific Ca2+/CaM-dependent kinase MFs. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-uniprot.txt Serine/threonine-protein kinase |
| GO:0005634 nucleus | EXP PMID:22869603 OsDMI3 is a novel component of abscisic acid signaling in th... | ACCEPT | Summary: EXP annotation citing PMID:22869603, the rice study that experimentally determined OsDMI3 subcellular localization. Directly supports nuclear localization. Reason: Directly supported by the cited experiment. Subcellular localization analysis in PMID:22869603 showed that OsDMI3 is located in the nucleus (as well as the cytoplasm and plasma membrane). Nuclear localization is the functionally critical compartment for decoding the nuclear Ca2+ spiking signal. Core localization. Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane |
| GO:0005737 cytoplasm | EXP PMID:22869603 OsDMI3 is a novel component of abscisic acid signaling in th... | ACCEPT | Summary: EXP annotation citing PMID:22869603. Directly supports cytoplasmic localization of OsDMI3 by experimental subcellular-localization analysis. Reason: Directly supported. The localization analysis in PMID:22869603 placed OsDMI3 in the cytoplasm in addition to the nucleus and plasma membrane. The cytoplasmic pool is consistent with the kinase's role in cytoplasmic ABA/H2O2 stress signaling. Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane |
| GO:0005886 plasma membrane | EXP PMID:22869603 OsDMI3 is a novel component of abscisic acid signaling in th... | ACCEPT | Summary: EXP annotation citing PMID:22869603. Directly supports plasma-membrane association of OsDMI3 by experimental subcellular-localization analysis. Reason: Directly supported. PMID:22869603 reported OsDMI3 at the plasma membrane (with nucleus and cytoplasm); UniProt classifies the cell-membrane pool as a peripheral membrane protein. Accept as a genuine, experimentally observed localization. Supporting Evidence: PMID:22869603 OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:17965173 Fungal symbiosis in rice requires an ortholog of a legume co... | NEW | Summary: OsCCaMK/OsDMI3 is genetically required for arbuscular mycorrhizal (AM) symbiosis in rice. This is the gene's genuine symbiotic process (replacing the incorrect "nodulation" keyword), demonstrated by Myc-defective Osccamk mutants and by the rescue-of-fungal- symbiosis phenotype in PMID:17965173. Reason: Rice does not nodulate, so the SPKW "nodulation" term (GO:0009877) is an organism- context error; the symbiotic biology the kinase truly enables in rice is AM symbiosis. OsCCaMK is the rice ortholog of the legume common-symbiosis gene and "Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene encoding a Ca2+/ calmodulin-dependent protein kinase" (PMID:17965173, the IMP reference). Osccamk loss-of-function mutants are Myc-defective (no cortex invasion / no arbuscule formation) and OsCCaMK is genetically required for AM accommodation [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. GO:0036377 "arbuscular mycorrhizal association" is the precise, non-obsolete process term and is the proposed replacement for the retired nodulation keyword. IMP is justified by the Osccamk mutant AM phenotype. Supporting Evidence: PMID:17965173 We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md Loss of rice CCAMK strongly impairs **arbuscular mycorrhizal colonization**, with fungal entry defects and failure of proper cortical colonization file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md CCaMK phosphorylates CYCLOPS and DELLA proteins help route signaling outputs toward symbiosis-specific transcriptional programs such as **RAM1** (AM) or **NIN** (nodulation in legumes) |
| GO:0005516 calmodulin binding | IBA GO_REF:0000033 | NEW | Summary: Calmodulin binding is an IBA annotation in the UniProt cross-references (GO_Central) for this protein and is mechanistically core: CCaMK/DMI3 has a dedicated calmodulin-binding region that, together with the EF-hands, makes its kinase activity Ca2+/calmodulin- dependent. Added here as it was not in the seeded GOA TSV but is a genuine MF. Reason: The "calcium/calmodulin-dependent protein kinase activity" MF (GO:0004683) implies calmodulin binding, and UniProt annotates an explicit calmodulin-binding region (residues 321-334). CCaMK/DMI3 proteins have an autoinhibitory/CaM-binding region whose occupancy by Ca2+-calmodulin regulates kinase output [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. This is a core regulatory molecular function and is supported by the IBA propagation across the CaMK group. Supporting Evidence: file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md CCaMK family proteins are regulated by **both Ca2+ and Ca2+/CaM** and contain a **CaM-binding/autoinhibitory domain** |
| GO:0009738 abscisic acid-activated signaling pathway | IMP PMID:22869603 OsDMI3 is a novel component of abscisic acid signaling in th... | NEW | Summary: OsCCaMK/OsDMI3 is a component of abscisic-acid signaling that induces antioxidant defense in rice leaves. This separable (non-symbiotic) function is directly demonstrated in PMID:22869603 and is not represented in the current GOA set. Reason: PMID:22869603 shows that OsDMI3 is required for ABA-induced increases in the activities of the antioxidant enzymes SOD and CAT, that ABA/H2O2/PEG induce OsDMI3 expression and activity, and that "OsDMI3 is an important component in ABA-induced antioxidant defense in rice." This is a genuine, experimentally supported biological process distinct from symbiosis. OsDMI3 acts upstream of OsMPK1 and phosphorylates OsRBOHB to potentiate ABA signaling [file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md]. GO:0009738 is the precise process term; IMP is justified by the OsDMI3 RNAi/mutant ABA phenotypes. Supporting Evidence: PMID:22869603 OsDMI3 is required for ABA-induced increases in the expression and the activities of superoxide dismutase (SOD) and catalase (CAT) PMID:22869603 OsDMI3 is an important component in ABA-induced antioxidant defense in rice file:ORYSJ/CCAMK/CCAMK-deep-research-falcon.md OsDMI3 phosphorylates **OsRBOHB** (NADPH oxidase) to promote **H2O2** production, potentiating **ABA signaling** and **drought stress tolerance** |
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Download this section (compressed HTML)Q: Does rice OsCCaMK have any cryptic role in interactions with diazotrophic (nitrogen-fixing) endophytes, or is its symbiotic function in rice strictly limited to arbuscular mycorrhization given the absence of a nodulation program?
Suggested experts: Haruko Imaizumi-Anraku
Q: Are the symbiotic (AM, nuclear, CYCLOPS-directed) and the abiotic-stress (ABA-ROS, cytoplasmic/membrane, OsRBOHB/OsMPK1-directed) activities of OsDMI3 mechanistically separable - e.g. governed by different Ca2+ signatures or different subcellular pools?
Suggested experts: Mingyi Jiang
Experiment: Quantify arbuscular mycorrhizal colonization (intraradical hyphae, arbuscule density) in Osccamk loss-of-function mutants versus wild type after inoculation with Rhizophagus irregularis, and test complementation with the wild-type OsCCaMK transgene.
Hypothesis: OsCCaMK is strictly required for AM symbiosis in rice (Myc-defective), confirming that the gene's true symbiotic process is arbuscular mycorrhizal association, not nodulation.
Type: AM colonization phenotyping with genetic complementation
Experiment: Map and mutate the EF-hand, CaM-binding and autophosphorylation determinants of OsCCaMK and assay, in parallel, AM colonization rescue and ABA-induced antioxidant-enzyme activation, to test whether the two output pathways use distinct regulatory inputs.
Hypothesis: Symbiotic and ABA-ROS outputs of OsDMI3 are separable functions controlled by distinct Ca2+/CaM regulatory states of the same kinase.
Type: structure-function mutagenesis with dual phenotypic readouts
Experiment: Test whether OsCCaMK directly phosphorylates OsRBOHB and CYCLOPS/IPD3 in vitro (recombinant kinase + substrate, Ca2+/CaM-dependence) and identify phosphosites by mass spectrometry.
Hypothesis: OsCCaMK directly phosphorylates CYCLOPS (symbiosis) and OsRBOHB (ABA-ROS), explaining its dual role through distinct substrates.
Type: in vitro kinase / phosphosite mapping
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Download this section (compressed HTML)The YAML description field was revised to keep it as a standalone biological summary. Project-specific curation framing moved here instead.
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