RAM1 (REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1) is a GRAS-domain transcriptional regulator of Medicago truncatula that acts in root cortical cells as a central regulator of the arbuscular mycorrhizal (AM) gene program. RAM1 transcription is induced in colonized cortical cells and is activated downstream of the common symbiosis signalling pathway by a CCaMK-CYCLOPS-DELLA complex acting on the RAM1 promoter, integrating symbiotic and gibberellin signals. RAM1 is required for the expression of arbuscule-associated genes, including the glycerol-3-phosphate acyltransferase RAM2 (a direct target), the half-ABC transporter STR, the exocyst subunit EXO70I, the phosphate transporter PT4 and the GRAS factor RAD1, and ectopic RAM1 expression is sufficient to induce several of these genes in the absence of the fungus. Through this program RAM1 drives the host lipid biosynthesis that feeds the fatty-acid-auxotrophic fungus and the formation of the periarbuscular interface. ram1 mutants form arbuscules arrested at the trunk stage with few or no branches and have strongly reduced colonization; an early hyphopodium defect reported for ram1-1 was not observed for ram1-3. RAM1 is also required for the pre-symbiotic transcriptional response to fungal Myc-LCO signals and for phosphate-mediated suppression of colonization, but is dispensable for rhizobial Nod factor signalling. RAM1 interacts with the GRAS proteins NSP2, RAD1 and TF80 and acts in the nucleus.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003700 DNA-binding transcription factor activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (PAINT) inference that GRAS proteins of this clade, including RAM1, act as DNA-binding transcription factors. RAM1 itself activates symbiosis genes; RAM2 is described as a direct RAM1 target (PMID:28596311) and ectopic RAM1 expression activates arbuscule genes without the fungus (PMID:27020747). Reason: Consistent with the experimental data on RAM1 and its Lotus/petunia orthologs, which act as central transcriptional regulators of AM genes. Direct sequence-specific DNA binding by RAM1 has not been shown for most targets (binding to STR and EXO70I promoters was not demonstrated), but RAM2 is reported as a direct target and the IBD node placement is not contradicted by target-specific evidence. Accepted as the best available molecular function term. Supporting Evidence: PMID:28596311 this lipid export requires the glycerol-3-phosphate acyltransferase RAM2, a direct target of RAM1 PMID:27020747 ectopic expression of RAM1 activates genes critical for arbuscule development in the absence of fungal symbionts |
| GO:0003700 DNA-binding transcription factor activity | IMP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | ACCEPT | Summary: Park et al. showed by transcript analysis of ram1-3 and ectopic RAM1 expression that RAM1 regulates EXO70I, STR and RAD1 expression. Reason: Loss- and gain-of-function data show RAM1 controls transcription of arbuscule genes; the curators read the full text (cached copy is abstract only). Mutant phenotypes alone do not demonstrate DNA binding, and the deep research notes RAM1 binding to STR/EXO70I promoters was not demonstrated in this study, but the transcription-factor function is supported overall (RAM2 as direct target, PMID:28596311) and the same term is placed by IBA. Accepted, deferring to the curator. Supporting Evidence: PMID:26511916 Transcript analysis of ram1-3 and ectopic expression of RAM1 indicate that RAM1 regulates expression of EXO70I and Stunted Arbuscule, two genes whose loss of function impacts arbuscule branching. file:MEDTR/RAM1/RAM1-deep-research-falcon.md Park *et al.* could **not** demonstrate MtRAM1 binding to STR or EXO70I upstream sequences |
| GO:0005515 protein binding | IPI PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | MODIFY | Summary: Interaction of RAM1 with the GRAS protein TF80 (G7J1L1), reported by Park et al. (yeast interaction assays per UniProt and deep research). Reason: Protein binding is uninformative. GRAS proteins function as hetero-oligomeric transcriptional complexes (e.g. NSP1-NSP2), and RAM1 interacts with several other GRAS proteins (NSP2, RAD1, TF80). The interaction is better captured as protein heterodimerization activity between GRAS transcription factors. The functional significance of the TF80 interaction is unknown. Proposed replacements: protein heterodimerization activity Supporting Evidence: file:MEDTR/RAM1/RAM1-deep-research-falcon.md MtRAM1 interacts with NSP2, RAD1 and TF80 in yeast interaction experiments |
| GO:0005515 protein binding | IPI PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | MODIFY | Summary: Interaction of RAM1 with RAD1 (G7JMM0), a GRAS transcription factor whose expression is itself RAM1-dependent and which is required for AM symbiosis. Reason: Protein binding is uninformative; the RAM1-RAD1 interaction is a GRAS-GRAS heteromeric interaction among AM transcriptional regulators and is better represented as protein heterodimerization activity. Proposed replacements: protein heterodimerization activity Supporting Evidence: PMID:26511916 Furthermore, RAM1 regulates expression of a transcription factor Required for Arbuscule Development (RAD1). file:MEDTR/RAM1/RAM1-deep-research-falcon.md MtRAM1 interacts with NSP2, RAD1 and TF80 in yeast interaction experiments |
| GO:0005515 protein binding | IPI PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | MODIFY | Summary: Interaction of RAM1 with NSP2 (Q5NE24), the GRAS protein shared between nodulation and mycorrhizal signalling; RAM1-NSP2 interaction was first reported by Gobbato et al. 2012 and associated with RAM2 promoter regulation. Reason: Protein binding is uninformative. The RAM1-NSP2 interaction parallels the NSP1-NSP2 GRAS heterocomplex of nodulation and is best captured as protein heterodimerization activity. Proposed replacements: protein heterodimerization activity Supporting Evidence: PMID:23122845 In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. file:MEDTR/RAM1/RAM1-deep-research-falcon.md earlier work associates RAM1 with the RAM2 promoter through NSP2 |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Nuclear site of action inferred phylogenetically for GRAS transcription regulators; consistent with IDA for RAM1 itself (PMID:23122845). Reason: Nucleus is the site of action of a transcriptional regulator; supported by experimental localization of RAM1 and of its GRAS partners. Supporting Evidence: PMID:23122845 Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. |
| GO:0005634 nucleus | IDA PMID:23122845 A GRAS-type transcription factor with a specific function in... | ACCEPT | Summary: Gobbato et al. 2012 report RAM1 in the nucleus (UniProt subcellular location cites this paper). The cached copy is abstract-only. Reason: Experimental localization from the gene's cloning paper; consistent with its transcriptional role. The deep research notes that native RAM1 localization in Medicago roots has limited support (heterologous/BiFC data), but the curator read the full text and the nuclear location is biologically coherent. Supporting Evidence: PMID:23122845 Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the UniProt subcellular location (Nucleus, from PMID:23122845). Reason: Redundant with the IDA annotation but correct. Supporting Evidence: PMID:23122845 Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapping of the UniProt subcellular location "Cytoplasm", itself a by-similarity statement transferred from Lotus RAM1 (homodimer observed in nucleus and cytoplasm). Reason: Cytoplasmic signal of the Lotus ortholog likely reflects transient or overexpression-based observation (homodimerization assays); the functional site of a transcriptional regulator is the nucleus. Not wrong, but not part of the core function. Supporting Evidence: file:MEDTR/RAM1/RAM1-deep-research-falcon.md MtRAM1 is expected to act principally **in plant nuclei** |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Transferred by sequence similarity from Lotus japonicus RAM1 (A0A145P7T2), reported to homodimerize in nucleus and cytoplasm. Reason: Reasonable ortholog transfer, but cytoplasmic presence is not where RAM1 exerts its function; retained as non-core. Supporting Evidence: file:MEDTR/RAM1/RAM1-deep-research-falcon.md MtRAM1 is expected to act principally **in plant nuclei** |
| GO:0006355 regulation of DNA-templated transcription | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of transcriptional regulation for GRAS family members; RAM1 appears among the experimental descendants. Reason: Correct; RAM1 regulates transcription of AM genes. Directionally, RAM1 acts as an activator (see proposed positive-regulation term on the IMP row). Supporting Evidence: PMID:27020747 ectopic expression of RAM1 activates genes critical for arbuscule development in the absence of fungal symbionts |
| GO:0006355 regulation of DNA-templated transcription | IMP PMID:23122845 A GRAS-type transcription factor with a specific function in... | ACCEPT | Summary: ram1 mutants fail to induce RAM2; RAM1 regulates RAM2 expression (Gobbato et al. 2012). Reason: Correct. The evidence shows RAM1 is required for, and sufficient to drive, induction of target genes (RAM2, STR, EXO70I, RAD1, lipid biosynthesis genes), so RAM1 acts as an activator; the directional positive-regulation term is proposed on the RNA polymerase II row and in core_functions, while this general term is kept consistent with the IBA. Supporting Evidence: PMID:23122845 RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. PMID:28596311 we demonstrate that it is required and sufficient for the induction of a lipid biosynthetic pathway that is expressed in plant cells accommodating fungal arbuscules |
| GO:0006357 regulation of transcription by RNA polymerase II | IMP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | MODIFY | Summary: RAM1 is required for expression of EXO70I, STR and RAD1 and ectopic expression induces them (Park et al. 2015). Reason: Correct in essence (protein-coding target genes, hence RNA polymerase II); RAM1 acts as an activator of these genes, so the positive-regulation child term is more precise. Proposed replacements: positive regulation of transcription by RNA polymerase II Supporting Evidence: PMID:26511916 Transcript analysis of ram1-3 and ectopic expression of RAM1 indicate that RAM1 regulates expression of EXO70I and Stunted Arbuscule, two genes whose loss of function impacts arbuscule branching. |
| GO:0007584 response to nutrient | IMP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | KEEP AS NON CORE | Summary: ram1-3 is insensitive to phosphate-mediated suppression of AM colonization (Park et al. 2015). Reason: The phenotype indicates RAM1 is needed for high phosphate to restrain the symbiosis, consistent with phosphate status feeding into RAM1-dependent transcription. However, the reduced colonization of ram1 mutants complicates interpretation, and the mechanism is unknown. Retained as a non-core, physiological role. Supporting Evidence: PMID:26511916 ram1-3 is also insensitive to phosphate-mediated regulation of the symbiosis. |
| GO:0009610 response to symbiotic fungus | IEP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | KEEP AS NON CORE | Summary: RAM1 transcription is induced in colonized root cortical cells. Reason: Expression-pattern evidence; correct as a description of RAM1 induction by AM fungi but its functional role is better captured by arbuscular mycorrhizal association. Supporting Evidence: PMID:26511916 RAM1 itself is induced in colonized root cortical cells |
| GO:0010247 detection of phosphate ion | IMP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | MARK AS OVER ANNOTATED | Summary: Based on the insensitivity of ram1-3 to phosphate-mediated regulation of the symbiosis. Reason: Detection of phosphate ion is the reception of the phosphate stimulus and its conversion to a signal. A GRAS transcriptional regulator acting downstream in the symbiotic program is not shown to perceive phosphate; the mutant phenotype indicates that the phosphate response requires RAM1 (necessity), not that RAM1 participates in detection. The broader response to nutrient annotation already captures this phenotype. Supporting Evidence: PMID:26511916 ram1-3 is also insensitive to phosphate-mediated regulation of the symbiosis. |
| GO:0036377 arbuscular mycorrhizal association | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT placement of AM association at the RAM1-ortholog node, grounded in experimental data on RAM1 itself. Reason: RAM1 orthologs are required for AM symbiosis in Medicago, Lotus and petunia; RAM1 is conserved in AM-host lineages, consistent with a node restricted to the RAM1 clade. Supporting Evidence: PMID:25971550 The lack of ATA, which represents the ortholog of Required For Arbuscular Mycorrhiza1 in Medicago truncatula, renders the interaction completely ineffective |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:23122845 A GRAS-type transcription factor with a specific function in... | ACCEPT | Summary: ram1 mutants are unable to be colonized by mycorrhizal fungi (Gobbato et al. 2012). Reason: Core process. RAM1 transcriptionally drives the host arbuscule program. Note that the hyphopodium defect reported here for ram1-1 was not reproduced for ram1-3 by Park et al. (PMID:26511916), who found the primary defect is arbuscule branching; the process annotation stands either way. Supporting Evidence: PMID:23122845 Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. PMID:23122845 RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:25971550 The Petunia GRAS Transcription Factor ATA/RAM1 Regulates Sym... | ACCEPT | Summary: The petunia ortholog ATA/RAM1 is the central regulator of AM-related genes and is required for arbuscule morphogenesis. Reason: The cached abstract describes mutants of the petunia ortholog, so for the Medicago protein this is strictly ortholog evidence (ISS-like) rather than a direct mutant phenotype. The full text was not available to check whether Medicago material was also used. The process is clearly correct for Medicago RAM1 (PMID:23122845, PMID:26511916), so the annotation is accepted, deferring to the curator on evidence code. Supporting Evidence: PMID:25971550 Atypical Arbuscule (ATA), that acts as the central regulator of AM-related genes and is required for the morphogenesis of arbuscules |
| GO:0036377 arbuscular mycorrhizal association | IMP PMID:26511916 Hyphal Branching during Arbuscule Development Requires Reduc... | ACCEPT | Summary: ram1-3 forms arbuscules arrested at the trunk stage; RAM1 is essential for arbuscule branching. Reason: Core process, directly supported by the mutant phenotype in Medicago. Supporting Evidence: PMID:26511916 RAM1 is not necessary to enable hyphopodium formation or hyphal entry into the root but is essential to support arbuscule branching |
Loading supporting content…
Download this section (compressed HTML)Q: Does RAM1 bind DNA directly and sequence-specifically, or does it activate targets such as RAM2, STR and EXO70I only as part of a GRAS heterocomplex (e.g. with NSP2 or RAD1) whose DNA-binding is supplied by a partner? This determines whether DNA-binding transcription factor activity or transcription coregulator activity is the correct molecular function.
Q: Is RAM1 required for hyphopodium formation (as reported for ram1-1) or only for arbuscule branching (as reported for ram1-3), and do the alleles or the growth conditions explain the discrepancy?
Q: Should the PMID:25971550 IMP annotation (a petunia ATA mutant study) be recoded as ISS/ISO for the Medicago protein?
Q: How does phosphate status act on RAM1-dependent transcription; is RAM1 regulated by PHR-SPX phosphate signalling directly?
Experiment: DAP-seq or ChIP-seq of RAM1 in mycorrhizal Medicago roots, together with EMSA of recombinant RAM1 alone and with NSP2/RAD1, to establish direct targets and whether RAM1 binds DNA autonomously.
Experiment: Native-promoter RAM1-fluorescent protein fusions in complemented ram1 roots to determine subcellular localization in arbusculated cortical cells.
Experiment: Side-by-side early-stage phenotyping of ram1-1, ram1-2 and ram1-3 under identical inoculation conditions to resolve the hyphopodium discrepancy.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)