CYCLOPS (also called IPD3, INTERACTING PROTEIN OF DMI3) is a plant-specific 518-residue nuclear protein of Lotus japonicus that acts as a phosphorylation-activated, sequence-specific DNA-binding transcriptional activator in the common symbiosis signalling pathway shared by root nodule symbiosis with rhizobia and arbuscular mycorrhiza (AM) with glomeromycotan fungi. It has no similarity to known protein families outside the CYCLOPS family; it carries an N-terminal negative regulatory region, two predicted nuclear localization signals (the C-terminal-proximal one shown to be functional), a central transactivation region and a C-terminal coiled-coil region that contributes to DNA binding. CYCLOPS forms a preassembled, stimulus-independent nuclear complex with the calcium- and calmodulin-dependent protein kinase CCaMK, which decodes symbiotic nuclear calcium oscillations; CCaMK phosphorylates CYCLOPS, and phosphorylation of two N-terminal serines (Ser50 and Ser154) is required for CYCLOPS activity. Phosphorylated CYCLOPS binds cis-regulatory CYCLOPS-response elements and transactivates symbiosis genes, including NODULE INCEPTION (NIN; initiating a NIN/NF-Y cascade for nodule organogenesis), the AP2/ERF transcription factor gene ERN1 (required for infection-thread formation), and, together with CCaMK and DELLA proteins, the GRAS transcription factor gene RAM1 that controls arbuscule branching. cyclops mutants are impaired in rhizobial infection-thread formation and in AM fungal entry of outer cortical cells and arbuscule development, while nodule primordia can still form; a phosphomimetic CYCLOPS is sufficient to trigger spontaneous nodule organogenesis without rhizobia or CCaMK. The function is conserved across angiosperms, as rice CYCLOPS is required for AM and complements Lotus cyclops mutants.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003700 DNA-binding transcription factor activity | IDA PMID:27020747 A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM... | MODIFY | Summary: CYCLOPS binds a cis element in the RAM1 promoter and, in a complex with CCaMK and DELLA, activates RAM1 transcription (PMID:27020747). Its DNA-binding transcriptional activator activity is independently shown for the NIN promoter (PMID:24528861) and the ERN1 promoter (PMID:28503742). Reason: The annotation is correct and represents the core molecular function, but all characterized CYCLOPS targets (RAM1, NIN, ERN1) are activated, and CYCLOPS has been shown to be a transcriptional activator with a mapped transactivation region. The more specific child term DNA-binding transcription activator activity (GO:0001216) captures this. The RNA polymerase II-specific variant is not used because the polymerase specificity has not been addressed experimentally for this plant protein. Proposed replacements: DNA-binding transcription activator activity Supporting Evidence: PMID:27020747 The corresponding proteins form a complex that activates RAM1 expression via binding of CYCLOPS to a cis element in the RAM1 promoter. PMID:24528861 We show that CYCLOPS, a direct phosphorylation substrate of CCaMK, is a DNA-binding transcriptional activator. |
| GO:0005515 protein binding | IPI PMID:19074278 CYCLOPS, a mediator of symbiotic intracellular accommodation... | MODIFY | Summary: The interacting partner A0AAR7 is L. japonicus CCaMK. Yano et al. showed direct CYCLOPS-CCaMK interaction by Y2H and BiFC in plant nuclei; the interaction requires kinase-active CCaMK and CYCLOPS is phosphorylated by CCaMK in vitro. Reason: Generic protein binding is uninformative. The cited paper directly supports interaction with a protein kinase (CCaMK), of which CYCLOPS is a phosphorylation substrate and with which it forms a preassembled nuclear complex. Protein kinase binding (GO:0019901) is the evidence-backed, more informative term. This binding is a mechanistic feature of the CCaMK-CYCLOPS module rather than CYCLOPS's core activity, which is transcriptional activation. Proposed replacements: protein kinase binding Supporting Evidence: PMID:19074278 We show that CYCLOPS specifically interacts with kinase-active CCaMK in planta and is phosphorylated by CCaMK in vitro, suggesting that CYCLOPS is a phosphorylation target of CCaMK. PMID:19074278 Strong fluorescence was observed in the nucleus when CYCLOPS and CCaMK fused to the C- and N-terminal half of YFP, respectively, were coexpressed |
| GO:0005634 nucleus | IDA PMID:19074278 CYCLOPS, a mediator of symbiotic intracellular accommodation... | ACCEPT | Summary: GFP-CYCLOPS was detected exclusively in the nucleus of N. benthamiana epidermal cells, deletion of the C-terminal-proximal NLS abolished nuclear restriction, and CYCLOPS colocalized with CCaMK in nuclei of L. japonicus root cells. Reason: Direct localization evidence with NLS mapping; the nucleus is where CYCLOPS acts as a transcription factor. Supporting Evidence: PMID:19074278 GFP-CYCLOPS and a C-terminal truncation lacking the coiled-coil motif (GFP-CYCLOPS 1–449) were exclusively detected in the cell nucleus |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic nucleus annotation from the CYCLOPS family signature and the UniProt subcellular location, consistent with experimental data. Reason: Consistent with direct experimental localization of CYCLOPS to the nucleus (PMID:19074278). Supporting Evidence: PMID:19074278 CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. |
| GO:0006355 regulation of DNA-templated transcription | IDA PMID:27020747 A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM... | MODIFY | Summary: CYCLOPS, in a complex with CCaMK and DELLA, activates RAM1 transcription by binding a cis element in the RAM1 promoter. Reason: Correct but less specific than the evidence warrants: the RAM1 data (and the NIN and ERN1 data) show positive regulation. Positive regulation of DNA-templated transcription (GO:0045893) is already annotated from PMID:28503742 and is the appropriate level. Proposed replacements: positive regulation of DNA-templated transcription Supporting Evidence: PMID:27020747 The corresponding proteins form a complex that activates RAM1 expression via binding of CYCLOPS to a cis element in the RAM1 promoter. |
| GO:0009877 nodulation | IMP PMID:19074278 CYCLOPS, a mediator of symbiotic intracellular accommodation... | ACCEPT | Summary: cyclops mutants form nodule primordia but no infection threads, and nodule development arrests prematurely; mutant plants are stunted under nitrogen-limiting conditions with Mesorhizobium loti. Complementation with genomic CYCLOPS restored wild-type nodulation. Subsequent work showed that phosphorylated CYCLOPS directly activates NIN and that a phosphomimetic CYCLOPS is sufficient to trigger nodule organogenesis (PMID:24528861), and that CYCLOPS activates ERN1, required for infection threads (PMID:28503742). Reason: CYCLOPS is a transcription factor that directly activates the nodulation regulators NIN and ERN1, so it does part of the work of the nodulation program (transcriptional induction of the symbiotic gene cascade) rather than merely being required for it. Yano et al. noted that CYCLOPS is dispensable for organogenesis when CCaMK is deregulated, but its infection role and the later gain-of-function data justify the process annotation. Supporting Evidence: PMID:19074278 Hairy root transformation of cyclops-3 with the genomic CYCLOPS sequence, including the native promoter, resulted in the development of WT-like nodules upon inoculation with M. loti PMID:24528861 A phosphomimetic version of CYCLOPS was sufficient to trigger root nodule organogenesis in the absence of rhizobia and CCaMK. PMID:28503742 We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells. |
| GO:0036377 arbuscular mycorrhizal association | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the CYCLOPS family signature. CYCLOPS orthologues are required for AM in Lotus and rice, and the family is conserved in AM-forming plants. Reason: The family-level mapping is well founded: loss of CYCLOPS blocks AM in both a legume and a monocot, and rice CYCLOPS complements Lotus cyclops mutants. Supporting Evidence: PMID:19074278 Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:19074278 CYCLOPS, a mediator of symbiotic intracellular accommodation... | ACCEPT | Summary: On cyclops roots AM fungal hyphae form aberrant swellings in epidermal and outer cortical cells and arbuscules are almost completely absent; complementation with CYCLOPS cDNA restores AM. Reason: Mutant phenotype with complementation; combined with the later demonstration that CYCLOPS directly activates RAM1 (PMID:27020747), this reflects direct participation via transcriptional activation of the AM program. Supporting Evidence: PMID:19074278 These data indicate that CYCLOPS is required for fungal infection of the outer cortical cell layers and for arbuscule development. |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:27020747 A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM... | ACCEPT | Summary: Epistasis analysis places RAM1 downstream of CYCLOPS: ectopic RAM1 expression restores arbuscule formation in cyclops mutants, and CYCLOPS binds the RAM1 promoter to activate it. Reason: Genetic and molecular evidence that CYCLOPS drives the AM arbuscule program through RAM1 activation. Supporting Evidence: PMID:27020747 Epistasis analysis places RAM1 downstream of CCaMK, CYCLOPS, and DELLA because ectopic expression of RAM1 restores arbuscule formation in cyclops mutants and in the presence of suppressive gibberellin. |
| GO:0042803 protein homodimerization activity | IPI PMID:19074278 CYCLOPS, a mediator of symbiotic intracellular accommodation... | KEEP AS NON CORE | Summary: Self-interaction was detected by Y2H (requiring residues 81-366) and by BiFC in planta for full-length CYCLOPS. Reason: Experimentally supported, but the functional significance of CYCLOPS self-association for DNA binding or transactivation has not been established; it is a structural property rather than the core activity. Supporting Evidence: PMID:19074278 Using CYCLOPS 1–259 as bait revealed that CYCLOPS forms homodimers, and that the region between residues 81–366 is necessary for self-interaction file:LOTJA/CYCLOPS/CYCLOPS-deep-research-falcon.md protein-interaction measurements also detected its association with CCaMK and CYCLOPS homodimerization |
| GO:0043565 sequence-specific DNA binding | IDA PMID:27020747 A CCaMK-CYCLOPS-DELLA Complex Activates Transcription of RAM... | ACCEPT | Summary: CYCLOPS binds a cis element in the RAM1 promoter. Reason: Direct binding evidence to a defined promoter element; consistent with the sequence- and phosphorylation-dependent binding to the NIN CYC-box (PMID:24528861). It is a component of the core transcription activator activity. Supporting Evidence: PMID:27020747 activates RAM1 expression via binding of CYCLOPS to a cis element in the RAM1 promoter PMID:24528861 CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner and transactivates the NODULE INCEPTION (NIN) gene. |
| GO:0043565 sequence-specific DNA binding | IDA PMID:28503742 The ERN1 transcription factor gene is a target of the CCaMK/... | ACCEPT | Summary: CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter. Reason: Direct DNA-protein interaction assay with a defined motif. Supporting Evidence: PMID:28503742 Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter |
| GO:0043565 sequence-specific DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the CYCLOPS family signature; consistent with experimental DNA-binding data for Lotus CYCLOPS. Reason: Supported by direct experimental evidence on this protein (PMID:24528861, PMID:28503742). Supporting Evidence: PMID:24528861 CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner |
| GO:0045893 positive regulation of DNA-templated transcription | IDA PMID:28503742 The ERN1 transcription factor gene is a target of the CCaMK/... | ACCEPT | Summary: CYCLOPS together with CCaMK positively regulates ERN1 transcription; it also transactivates NIN (PMID:24528861) and RAM1 (PMID:27020747). Reason: Direct transactivation evidence at defined promoter elements; this is the core biological role of CYCLOPS at the molecular level. Supporting Evidence: PMID:28503742 CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. file:LOTJA/CYCLOPS/CYCLOPS-deep-research-falcon.md CYCLOPS also directly regulates the Lotus ***ERN1*** promoter |
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Download this section (compressed HTML)Q: Is protein kinase binding (to CCaMK) the right way to capture the CCaMK-CYCLOPS relationship, or should it be modelled on the kinase side (CCaMK has_input CYCLOPS) in a GO-CAM of the common symbiosis pathway?
Q: How does a single CCaMK-CYCLOPS module select nodulation-specific (NIN, ERN1) versus AM-specific (RAM1) or shared (CBP1) target promoters; which partner proteins (e.g. DELLA) and promoter contexts determine specificity?
Q: What is the function, if any, of CYCLOPS homodimerization in DNA binding or transactivation?
Experiment: Genome-wide CYCLOPS binding (ChIP-seq or DAP-seq with wild-type versus phosphomimetic CYCLOPS-DD) in Lotus roots inoculated with rhizobia or AM fungi, to define the direct target repertoire for each symbiosis.
Experiment: Mutational analysis of the coiled-coil/self-interaction region combined with EMSA and transactivation assays to test whether dimerization is required for CYC-box binding.
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