id: F7J0M4 gene_symbol: RGA4 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:39947 label: Oryza sativa Japonica Group description: >- RGA4 (Os11gRGA4, SasRGA4) is a 996-residue coiled-coil, nucleotide-binding (NB-ARC), leucine-rich repeat (CC-NB-LRR, CNL) immune receptor of rice that, together with the adjacent and inversely oriented CNL gene RGA5, constitutes the Pia / Pi-CO39 blast resistance locus. The two proteins act as an obligate sensor/executor heteropair conferring recognition of the unrelated Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39. RGA4 is the executor: it is an intrinsically (constitutively) active inducer of hypersensitive-response cell death, and this activity is held in check by RGA5, with which it forms hetero-complexes through its N-terminal coiled-coil domain; RGA4 also self-associates. RGA4 itself does not bind the effectors. Binding of AVR-Pia or AVR1-CO39 to the integrated heavy-metal-associated (HMA/RATX1) domain of RGA5 relieves the repression and RGA4 triggers cell death and resistance. RGA4 activity requires an intact P-loop (K209) in its NB-ARC domain, and its autoactivity depends on a degenerate MHD motif (TYG) in the ARC2 subdomain. RGA4 resides mainly in the cytosol and does not relocalise to the nucleus on effector recognition. When ectopically activated it also restricts Xanthomonas oryzae, showing that its immune output is not pathogen-specific. alternative_records: - id: F7J0M6 relationship: REFERENCE_PROTEOME_LOCUS_COUNTERPART reference_proteome: UP000059680 reviewed_on: '2026-10-19' reason: Retain F7J0M4 as the primary Swiss-Prot accession. Both reviewed records represent RGA4 in different rice cultivars, and the reference entry is the leading full-length match. UniProt contrasts AVR-Pia recognition in Aichi asahi/Sasanishiki with its absence in Nipponbare. Record this as a reference-cultivar locus counterpart, not equivalent resistance function; loss of recognition by the RGA4/RGA5 system does not establish that RGA4 alone is inactive. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 48,896 sequences across UP000059680 at E <= 1e-5 and sensitivity 7.5, returning 358 hits. Every returned sequence was globally aligned using BLOSUM62 with gap-open -10 and extension -0.5. Scores and sequences for all 358 returned candidates are retained, with 10 detailed alignment files. The heuristic search does not establish genomic absence or unique locus identity. supported_by: - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'target_sequence_count: 48896' - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'candidate_count: 358' - type: SEQUENCE_COMPARISON description: 'F7J0M6: 951 identities across 1017 global-alignment columns (93.5103% identity including gaps), 42 substitutions and 24 gap columns. Query coverage is 99.6988%; target coverage is 97.929%. Sequence similarity is interpreted in the manual rationale. Identity over paired residues is 95.7704% (951/993); gap columns are excluded from this denominator.' supported_by: - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: |- query_length: 996 target_length: 1014 alignment_columns: 1017 identities: 951 mismatches: 42 gap_columns: 24 identity_percent: 93.5103 query_coverage_percent: 99.6988 target_coverage_percent: 97.929 - type: PROTEOME_MEMBERSHIP description: The primary accession is outside every searched exact-taxon reference proteome. Current UniProt records place F7J0M6 in UP000059680 and give it the same taxon as the primary. supported_by: - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'alternative_in_reference_proteome: true' - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'same_taxon: true' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 09-DEC-2015, entry version 17.: no Proteomes cross-reference; 12-AUG-2020, entry version 38.: no Proteomes cross-reference; 27-NOV-2024, entry version 48.: 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:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: |- entry_date: 09-DEC-2015, entry version 17. proteomes: [] - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: |- entry_date: 12-AUG-2020, entry version 38. proteomes: [] - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: |- entry_date: 27-NOV-2024, entry version 48. proteomes: [] - type: CURATION_CONTEXT description: The reviewed reference entry explicitly contrasts cultivar-dependent recognition of AVR-Pia. This concerns the resistance system and does not prove that RGA4 alone is inactive. supported_by: - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: cultivars Nipponbare, Mokoto and Hitomebore - type: LOCUS_MAPPING description: The reviewed reference record F7J0M6 assigns RGA4 to Os11g0225100 / LOC_Os11g11790. Both F7J0M4 and F7J0M6 use the RGA4 name attributed to PMID:21251109, the study of the chromosome-11 Pia locus across rice cultivars. Together with the leading full-length sequence match and the study's identification of the Sasanishiki Os11gRGA4 allele, this supports a curated locus correspondence. This is not an independent genomic coordinate alignment and does not establish equivalent resistance activity. supported_by: - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'value: Os11g0225100' - reference_id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml supporting_text: 'value: LOC_Os11g11790' - reference_id: PMID:21251109 supporting_text: |- The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants existing_annotations: - term: id: GO:0002758 label: innate immune response-activating signaling pathway evidence_type: IDA original_reference_id: PMID:23548743 qualifier: involved_in review: summary: >- RGA4 and RGA5 together are required for recognition of AVR1-CO39 and AVR-Pia and for triggering the immune response. Later work showed RGA4 is the component that generates the immune/cell-death signal once RGA5 repression is lifted. action: ACCEPT reason: >- RGA4 is the signalling executor of the RGA4/RGA5 pair, so it directly participates in the effector-triggered signalling pathway that activates innate immunity. GO has no host-side effector-triggered immunity signalling term, so this is the appropriate process term. supported_by: - reference_id: PMID:23548743 supporting_text: Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. - reference_id: PMID:25024433 supporting_text: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor. - term: id: GO:0002758 label: innate immune response-activating signaling pathway evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in supporting_entities: - ARBA:ARBA00034398 review: summary: >- ARBA machine-learning prediction for CNL proteins; consistent with the experimental IDA annotation for the same term. action: ACCEPT reason: >- Correct and experimentally corroborated for RGA4 as the signalling executor of the pair. supported_by: - reference_id: PMID:25024433 supporting_text: These results support the conclusion that cell death signaling is activated exclusively by RGA4 and that AVR-Pia binding does not lead to RGA5 activation but solely to RGA4 de-repression. - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:25024433 qualifier: enables supporting_entities: - UniProtKB:F7J0N2 review: summary: >- RGA4 co-immunoprecipitates with RGA5 in rice protoplasts and N. benthamiana, and the CC domains of the two proteins interact in yeast two-hybrid assays. RGA4 also self-associates. action: MODIFY reason: >- The interaction is real but "protein binding" is uninformative. The interaction is a CC-domain-mediated hetero-complex with RGA5 (and a homo-complex with RGA4 itself), better captured by protein heterodimerization activity, with homodimerization as a secondary term. Dimer stoichiometry is not formally established, so these remain approximations of "hetero-/homo-complex formation". proposed_replacement_terms: - id: GO:0046982 label: protein heterodimerization activity - id: GO:0042803 label: protein homodimerization activity supported_by: - reference_id: PMID:25024433 supporting_text: RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. - reference_id: PMID:25024433 supporting_text: CC-RGA4 1–130 and CC-RGA5 1–133 showed homo- and hetero-interactions - term: id: GO:0005737 label: cytoplasm evidence_type: IDA original_reference_id: PMID:25024433 qualifier: located_in review: summary: >- RGA4:GFP is observed mainly in the cytosol of rice protoplasts, with little or no nuclear signal. action: ACCEPT reason: >- Directly observed; the cytosol annotation from the same paper is the more specific term, but cytoplasm is correct. supported_by: - reference_id: PMID:25024433 supporting_text: Green fluorescence of RGA4:GFP was observed mainly in the cytosol labeled by mCherry - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0086 review: summary: >- Mapped from the UniProt subcellular location (Cytoplasm), which is itself based on PMID:25024433. action: ACCEPT reason: Consistent with the experimental localisation data. supported_by: - reference_id: PMID:25024433 supporting_text: These results suggest that RGA4 and RGA5 localize to the cytosol and are excluded from the nucleus. - term: id: GO:0005829 label: cytosol evidence_type: EXP original_reference_id: PMID:25024433 qualifier: located_in review: summary: >- RGA4 co-localises with a cytosolic mCherry marker in rice protoplasts and is not relocalised to the nucleus upon AVR-Pia recognition. action: ACCEPT reason: Most specific localisation supported by direct experimental data. supported_by: - reference_id: PMID:25024433 supporting_text: Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. - term: id: GO:0006952 label: defense response evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR044974 review: summary: >- InterPro2GO mapping from the plant disease resistance protein family (IPR044974). Correct but very general. action: MARK_AS_OVER_ANNOTATED reason: >- True but uninformative; RGA4 already carries the more specific plant-type hypersensitive response and innate immune signalling terms, and a defense response to fungus annotation is proposed. supported_by: - reference_id: PMID:25024433 supporting_text: RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae - term: id: GO:0009626 label: plant-type hypersensitive response evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in supporting_entities: - ARBA:ARBA00091908 review: summary: >- ARBA prediction; strongly corroborated by experimental data showing RGA4 is the executor of HR-like cell death. action: ACCEPT reason: Correct and core for RGA4. supported_by: - reference_id: PMID:25024433 supporting_text: Taken together, these results suggest that RGA4 acts as an HR executer which is regulated in its activity by RGA5. - term: id: GO:0009626 label: plant-type hypersensitive response evidence_type: IMP original_reference_id: PMID:23548743 qualifier: involved_in review: summary: >- Loss of RGA4 (and RGA5) abolishes AVR1-CO39/AVR-Pia-triggered resistance. The full text was not accessible; the abstract reports that both genes are required for effector recognition. action: ACCEPT reason: >- Consistent with the later demonstration that RGA4 is the HR executor; deferred to the curator who read the full text. supported_by: - reference_id: PMID:23548743 supporting_text: RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. - reference_id: PMID:25024433 supporting_text: RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. - term: id: GO:0009626 label: plant-type hypersensitive response evidence_type: IMP original_reference_id: PMID:25024433 qualifier: involved_in review: summary: >- RGA4 P-loop (K209R) and MHD-restoring/TYD mutants lose cell-death activity both alone and in the presence of RGA5 plus AVR-Pia; RGA5 silencing triggers cell death that depends entirely on RGA4. action: ACCEPT reason: >- Core function. RGA4 itself generates the HR cell death signal; this is the key executor annotation that distinguishes RGA4 from its sensor partner RGA5, which has no cell-death-inducing activity. supported_by: - reference_id: PMID:25024433 supporting_text: Thus, an intact RGA4 nucleotide-binding pocket is required for cell death induction by RGA4, either on its own or in the presence of RGA5 and AVR-Pia. - reference_id: PMID:25024433 supporting_text: In the rga4 loss-of-function-mutant line Sas1493 (Okuyama et al , 2011 ), RGA5 depletion had no effect indicating that the cell death induced by RGA5 depletion relies entirely on RGA4. - term: id: GO:0042742 label: defense response to bacterium evidence_type: IDA original_reference_id: PMID:23548743 qualifier: involved_in review: summary: >- The cached abstract of PMID:23548743 only addresses recognition of the fungal effectors AVR-Pia and AVR1-CO39, and full text could not be retrieved. Bacterial (Xanthomonas oryzae) resistance mediated by the RGA4/RGA5 pair was shown later, in engineered systems (PMID:27289079). action: MARK_AS_OVER_ANNOTATED reason: >- Not removed, because the supporting full text was not available to check. Biologically, RGA4 activation can restrict Xanthomonas oryzae, but only when AVR1-CO39 or an NLR is artificially induced by designer TAL effectors. There is no natural bacterial recognition specificity, so a bacterial defense term overstates RGA4's biological role. The same call is made for the sensor RGA5. The reference attribution may be worth rechecking against PMID:27289079. additional_reference_ids: - PMID:27289079 supported_by: - reference_id: PMID:27289079 supporting_text: This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. - term: id: GO:0042742 label: defense response to bacterium evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in supporting_entities: - ARBA:ARBA00028625 review: summary: >- ARBA prediction from the CNL family, many of whose characterised members recognise bacterial effectors. action: MARK_AS_OVER_ANNOTATED reason: >- The only support is engineered Xanthomonas resistance through the RGA4/RGA5 pair. The natural function of RGA4 is resistance to the blast fungus, so this is an over-annotation (consistent with the RGA5 review). supported_by: - reference_id: PMID:27289079 supporting_text: demonstrating that NLR-triggered immune responses efficiently control Xoo - term: id: GO:0043531 label: ADP binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR002182 review: summary: >- InterPro2GO mapping from the NB-ARC domain. Nucleotide binding has not been measured for RGA4, but the P-loop lysine K209 is required for all RGA4 activity, implying a functional nucleotide-binding pocket. action: ACCEPT reason: >- The NB-ARC ADP-binding mapping is the standard family-level representation of the nucleotide-dependent switch in NLRs, and RGA4 is the partner in which that pocket is functionally required (unlike RGA5). RGA4's degenerate MHD motif is thought to keep it out of the ADP-bound, resting state. ATP binding may therefore describe the active form better, but neither nucleotide has been measured. supported_by: - reference_id: PMID:25024433 supporting_text: This finding suggests that RGA4 activity is likely to be dependent on nucleotide binding and exchange while RGA5 activity is not. - reference_id: file:ORYSJ/RGA4/RGA4-deep-research-falcon.md supporting_text: eliminated its spontaneous and AVR-Pia-associated cell-death activity - term: id: GO:0062207 label: regulation of pattern recognition receptor signaling pathway evidence_type: EXP original_reference_id: PMID:25024433 qualifier: involved_in review: summary: >- PHI-base EXP annotation. PMID:25024433 (full text read) concerns effector recognition and cell-death activation by the intracellular CNL pair RGA4/RGA5. It contains no experiments on pattern recognition receptors or PRR signalling, which the paper explicitly distinguishes as the separate first layer of immunity. action: MODIFY reason: >- The term does not describe what RGA4 does. RGA4 is not a regulator of PRR (pattern-triggered) signalling. It is the executor of effector-triggered NLR signalling and HR. The essence of the annotation (an immune signalling role shown in this paper) is better captured by innate immune response-activating signaling pathway. That would also keep it distinct from the sensor RGA5, whose role in the same paper is repression of RGA4. proposed_replacement_terms: - id: GO:0002758 label: innate immune response-activating signaling pathway supported_by: - reference_id: PMID:25024433 supporting_text: The first layer of defense relies on the recognition of conserved microbe-associated molecular patterns (MAMPs) by the so-called pattern recognition receptors (PRRs). - reference_id: PMID:25024433 supporting_text: These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants - term: id: GO:0050832 label: defense response to fungus evidence_type: IMP original_reference_id: PMID:25024433 review: summary: >- RGA4 is required for Pia / Pi-CO39 resistance to Magnaporthe oryzae carrying AVR-Pia or AVR1-CO39. RGA4 loss-of-function mutants and P-loop or MHD mutants lose resistance or cell-death activity. No fungal defense term is present in GOA. action: NEW reason: >- Participation test passed. RGA4 is not a substrate or a passive requirement: it is the component that itself produces the immune and cell-death signal that restricts the fungus, while RGA5 does the sensing. This is the natural, primary biological context of RGA4, and it is better supported than the bacterial defense terms already in GOA. supported_by: - reference_id: PMID:25024433 supporting_text: We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae - reference_id: PMID:21251109 supporting_text: suggesting that SasRGA4 is necessary for Pia function core_functions: - description: >- Executor CC-NB-LRR of the RGA4/RGA5 heteropair. RGA4 is an intrinsically active, P-loop-dependent inducer of hypersensitive-response cell death and immune signalling in the cytosol. It is held inactive by RGA5 and released when AVR-Pia or AVR1-CO39 binds the RGA5 HMA domain. RGA4 does not bind the effectors itself. molecular_function: id: GO:0043531 label: ADP binding directly_involved_in: - id: GO:0009626 label: plant-type hypersensitive response - id: GO:0002758 label: innate immune response-activating signaling pathway - id: GO:0050832 label: defense response to fungus locations: - id: GO:0005829 label: cytosol supported_by: - reference_id: PMID:25024433 supporting_text: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor. - reference_id: PMID:25024433 supporting_text: Thus, an intact RGA4 nucleotide-binding pocket is required for cell death induction by RGA4, either on its own or in the presence of RGA5 and AVR-Pia. - reference_id: file:ORYSJ/RGA4/RGA4-deep-research-falcon.md supporting_text: RGA4 is the signaling and cell-death executor restrained by RGA5. - description: >- Forms CC-domain-mediated hetero-complexes with its sensor partner RGA5, through which RGA5 represses RGA4 in the absence of effector, and homo-complexes with itself. Effector binding to RGA5 does not dissociate the hetero-complex. molecular_function: id: GO:0046982 label: protein heterodimerization activity locations: - id: GO:0005829 label: cytosol supported_by: - reference_id: PMID:25024433 supporting_text: RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. - reference_id: PMID:25024433 supporting_text: This indicates that RGA4–RGA5 hetero-complexes still exist in the presence of AVR-Pia. suggested_questions: - question: >- GOA currently gives RGA4 (executor) and RGA5 (sensor) an almost identical set of annotations, drawn from the same two papers. Should RGA5's role be captured as negative regulation of RGA4-driven HR/immune signalling plus effector binding (innate immune receptor activity), and RGA4's as the positive executor of HR, so that the division of labour is machine-readable? - question: >- Does activated RGA4 form an oligomeric resistosome with Ca2+-permeable channel activity, as shown for other CC-NLRs such as ZAR1 and Sr35? If so, what is the appropriate executor molecular function term? - question: >- Is the defense response to bacterium IDA attributed to PMID:23548743 intended to reference PMID:27289079, the paper that tests Xanthomonas oryzae resistance? suggested_experiments: - description: >- Purify full-length or CC-domain RGA4, test oligomerisation and membrane association after RGA5 removal or effector addition, and measure Ca2+-permeable channel activity (for example by electrophysiology in Xenopus oocytes or HEK cells) to define the executor biochemistry. - description: >- Measure nucleotide binding of RGA4 (wild type, K209R and the MHD-restored and TYD variants) to determine whether its autoactive state is ATP-bound, which would decide between ADP binding and ATP binding as the correct molecular function term. references: - id: file:ORYSJ/RGA4/RGA4-record-evidence.yaml title: Compact archived record and sequence evidence for F7J0M4 - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms 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:0000117 title: Electronic Gene Ontology annotations created by ARBA machine learning models findings: [] - id: PMID:23548743 title: The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- PubMed-verified; abstract only (PMC full text not retrievable). It establishes the pair's dual specificity and that the effectors bind RGA5, not RGA4. Its abstract mentions no bacterial work, although GOA cites it for defense response to bacterium. - id: PMID:25024433 title: The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text read. The key paper establishing RGA4 as the autoactive HR executor repressed by RGA5, together with the P-loop/MHD requirements, the CC-mediated complexes and cytosolic localisation. - id: PMID:21251109 title: A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Gene isolation; RGA4 necessary but not sufficient for Pia. - id: PMID:27289079 title: Ectopic activation of the rice NLR heteropair RGA4/RGA5 confers resistance to bacterial blight and bacterial leaf streak diseases. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- Abstract only. Engineered (TAL-effector-induced) activation of AVR1-CO39 or an autoactive NLR confers Xanthomonas oryzae resistance; this supports the bacterial defense terms only as non-core. - id: file:ORYSJ/RGA4/RGA4-deep-research-falcon.md title: Deep research report on RGA4 (falcon) findings: []