id: Q00363 gene_symbol: AVR4 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:5499 label: Fulvia fulva description: >- AVR4 (race-specific elicitor A4; Cf-Avr4) is a small, cysteine-rich effector protein secreted by the biotrophic tomato leaf mould fungus Fulvia fulva (Cladosporium fulvum, Passalora fulva). It is made as a 135-residue precursor with a signal peptide and propeptide, and the mature protein carries an invertebrate-type chitin-binding domain (CBM14) stabilised by disulfide bonds. AVR4 is expressed during colonisation of the tomato leaf apoplast, where it binds chitin in the cell walls of the fungus's own hyphae. Binding is specific for chitin (no binding to other cell wall polysaccharides), requires chito-oligomers of at least three GlcNAc units, and occurs with micromolar affinity and roughly 1:1 stoichiometry to chitohexaose; the crystal structure of the AVR4-(GlcNAc)6 complex shows an extended binding surface centred on Trp100 and Asp102. By coating wall chitin, AVR4 shields hyphae from hydrolysis by plant chitinases, a non-enzymatic counter-defensive activity that requires chitin binding and contributes to full virulence (silencing of Avr4 reduces virulence on tomato, and heterologous expression increases susceptibility to other fungi with exposed wall chitin). AVR4 is also an avirulence determinant: tomato plants carrying the receptor-like protein Cf-4 recognise extracellular AVR4 and mount a hypersensitive response. Recognition depends on a stable, protease-resistant fold rather than on the chitin-binding residues, and Cf-4-virulent fungal strains typically carry disulfide-disrupting substitutions that make AVR4 protease-sensitive while preserving chitin binding. alternative_records: - id: L7YE01 relationship: REFERENCE_PROTEOME_EQUIVALENT reference_proteome: UP000756132 reviewed_on: '2026-10-19' reason: Retain Q00363 as the primary Swiss-Prot accession. The reference Avr4 sequence is exactly identical across all 135 residues and is the only significant hit. This supports equivalent protein representation while retaining the reviewed accession and its source-race-specific evidence. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 14,687 sequences across UP000756132 at E <= 1e-5 and sensitivity 7.5, returning 1 hits. Every returned sequence was globally aligned using BLOSUM62 with gap-open -10 and extension -0.5. Scores and sequences for all 1 returned candidates are retained, with 1 detailed alignment files. The heuristic search does not establish genomic absence or unique locus identity. supported_by: - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: 'target_sequence_count: 14687' - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: 'candidate_count: 1' - type: SEQUENCE_COMPARISON description: 'L7YE01: 135 identities across 135 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% (135/135); gap columns are excluded from this denominator.' supported_by: - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: |- query_length: 135 target_length: 135 alignment_columns: 135 identities: 135 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 L7YE01 in UP000756132 and give it the same taxon as the primary. supported_by: - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: 'alternative_in_reference_proteome: true' - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: 'same_taxon: true' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 09-DEC-2015, entry version 67.: no Proteomes cross-reference; 02-DEC-2020, entry version 81.: no Proteomes cross-reference; 27-MAR-2024, entry version 88.: 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:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: |- entry_date: 09-DEC-2015, entry version 67. proteomes: [] - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: |- entry_date: 02-DEC-2020, entry version 81. proteomes: [] - reference_id: file:FULFL/AVR4/AVR4-record-evidence.yaml supporting_text: |- entry_date: 27-MAR-2024, entry version 88. proteomes: [] existing_annotations: - term: id: GO:0005576 label: extracellular region evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: located_in supporting_entities: - InterPro:IPR002557 review: summary: >- AVR4 is a secreted protein (signal peptide plus propeptide) that is found in tomato apoplastic fluid and bound to fungal cell walls during infection. Extracellular region is correct, if general. action: ACCEPT reason: >- Secretion and extracellular action are well established. More specific locations (host apoplast; the fungal cell wall to which AVR4 binds) are proposed as NEW annotations below, but the general term is not wrong. supported_by: - reference_id: PMID:17849712 supporting_text: The Avr4 protein belongs to a set of effectors that is secreted by C. fulvum during infection - reference_id: PMID:12736265 supporting_text: The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. - term: id: GO:0008061 label: chitin binding evidence_type: EXP original_reference_id: PMID:30148881 qualifier: enables review: summary: >- Hurlburt et al. solved the 1.95 A crystal structure of AVR4 bound to (GlcNAc)6 and measured binding by ITC, identifying residues (W100, D102, Q69, K84) required for affinity. This is the core molecular function. action: ACCEPT reason: >- Direct structural and biophysical evidence on the target protein, confirmed by earlier independent studies (specific binding to chitin and chitin oligomers of at least three residues). supported_by: - reference_id: PMID:30148881 supporting_text: Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. - reference_id: PMID:17153926 supporting_text: We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. - term: id: GO:0008061 label: chitin binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR002557 - InterPro:IPR036508 review: summary: >- InterPro2GO mapping from the CBM14 chitin-binding domain. Correct and experimentally confirmed for this protein. action: ACCEPT reason: >- The domain-based inference agrees with the experimental evidence (PMID:30148881, PMID:17153926, PMID:12736265). supported_by: - reference_id: PMID:12736265 supporting_text: Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. - term: id: GO:0080185 label: effector-mediated activation of plant hypersensitive response by symbiont evidence_type: EXP original_reference_id: PMID:10998185 qualifier: involved_in review: summary: >- Laugé et al. injected purified secreted C. fulvum proteins, including the race-specific elicitor AVR4, into tomato lines and recorded HR-associated recognition. AVR4 triggers an HR in Cf-4 plants. action: KEEP_AS_NON_CORE reason: >- HR induction by AVR4 is real and is the standard GO representation of an avirulence (Avr) protein, but it is a consequence of host recognition by Cf-4 that is genetically and structurally separable from AVR4's intrinsic function (chitin shielding). It is therefore retained as non-core. supported_by: - reference_id: PMID:10998185 supporting_text: We found that HR-associated recognition of one or more of these proteins, in addition to recognition of the race-specific elicitors AVR4 and AVR9 of C. fulvum, occurs among Lycopersicon species. - reference_id: PMID:30148881 supporting_text: Taken together, these results indicate that mutations in residues within the ChBD that decrease or abolish the protein’s affinity for (GlcNAc)6 do not individually affect recognition by Cf-4 if they do not perturb the stability of the protein. - term: id: GO:0080185 label: effector-mediated activation of plant hypersensitive response by symbiont evidence_type: EXP original_reference_id: PMID:16167771 qualifier: involved_in review: summary: >- Kruijt et al. used AVR4 elicitor protein to identify AVR4-responsive wild tomato plants and Cf-4 homologues (Hcr9-Avr4s) mediating AVR4 recognition in agroinfiltration assays. action: KEEP_AS_NON_CORE reason: >- As for the PMID:10998185 row: AVR4-triggered, Cf-4-dependent HR is well supported, but represents the host recognition outcome, not the effector's intrinsic function. supported_by: - reference_id: PMID:16167771 supporting_text: Plants responsive to the Avr4 and Avr9 elicitor proteins were identified throughout the genus Lycopersicon. - term: id: GO:0140320 label: PAMP receptor decoy activity evidence_type: EXP original_reference_id: PMID:30148881 qualifier: enables review: summary: >- GO:0140320 is defined as binding and sequestering PAMP ligands to prevent them from binding and activating host PAMP receptors (the ECP6/LysM-effector mechanism). The cited paper (full text read) shows chitin binding and protection of fungal hyphae from chitinases, but contains no assay of PAMP sequestration, competition with a host chitin receptor, or suppression of chitin-triggered signalling by AVR4. action: MARK_AS_OVER_ANNOTATED reason: >- AVR4's demonstrated mechanism is shielding of wall-bound chitin from hydrolytic host chitinases, not sequestration of soluble chitin oligomers away from receptors. The authors themselves propose that AVR4 rests on the surface of chitin microfibrils as a protective layer against endochitinases, and they explicitly note uncertainty about whether binding to free chito-oligosaccharides is biologically relevant. Reduced release of chitin fragments could plausibly follow from wall shielding, but this has not been shown, so the decoy term over-interprets the evidence. The molecular activity is fully captured by chitin binding (GO:0008061). supported_by: - reference_id: PMID:30148881 supporting_text: Since, Avr4 binds (GlcNAc)6 in a 1:1 stoichiometric ratio, it is plausible that the protein rests as a monomer on the solvent-exposed surface of these microfibrils, thus creating a protective layer against endochitinases. - reference_id: PMID:30148881 supporting_text: If the case, it suggests that Avr4 may interact differently with free in solution chito-oligosaccharides as compared with chitin fixed in microfibrils. - term: id: GO:0140403 label: effector-mediated suppression of host innate immune response evidence_type: IMP original_reference_id: PMID:30148881 qualifier: involved_in review: summary: >- The IMP evidence is the in vitro protection assay: wild-type AVR4 protects Trichoderma viride germlings from plant chitinases plus beta-1,3-glucanases, whereas non-binding mutants (W100A, D102A) fail to protect. This shows that AVR4 counters an antifungal host defence effector (chitinase), not that it suppresses a host immune response. action: MODIFY reason: >- AVR4 does not suppress or dampen the host response; chitinases are still produced and active. Instead AVR4 makes the fungus resistant to a host innate immune effector with direct antifungal activity. GO:0141177 (symbiont-mediated evasion of recognition by host innate immune effector; "mitigates the effects of" host effectors such as antimicrobial peptides and complement that have direct activity against the symbiont) describes this precisely. The counter-defensive role is further supported by reduced virulence of Avr4-silenced C. fulvum on tomato (PMID:17849712). proposed_replacement_terms: - id: GO:0141177 label: symbiont-mediated evasion of recognition by host innate immune effector supported_by: - reference_id: PMID:30148881 supporting_text: Collectively, results from the protection assays show that mutations in the ChBD of CfAvr4 that decrease or abolish its affinity for (GlcNAc)6 also reduce the protein’s ability to protect fungal germlings against chitinases and thus perform its biological function. - reference_id: PMID:17153926 supporting_text: In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases - reference_id: PMID:17849712 supporting_text: Finally, we demonstrate that silencing of the Avr4 gene in C. fulvum decreases its virulence on tomato. - term: id: GO:0140404 label: effector-mediated perturbation of host innate immune response by symbiont evidence_type: EXP original_reference_id: PMID:23716655 qualifier: involved_in review: summary: >- Liebrand et al. (abstract only) used AVR4 as the ligand of Cf-4 to show that SOBIR1/EVR is required for the Cf-4-mediated hypersensitive response and immunity. AVR4 here acts as the trigger of host immunity. action: KEEP_AS_NON_CORE reason: >- The parent "perturbation" term covers activation, so it is not wrong; the experiment reflects Cf-4 recognition of AVR4, which is more specifically captured by the existing GO:0080185 rows. Retained as non-core because the avirulence role is a host recognition outcome rather than AVR4's intrinsic function. Full text not inspected; deferring to the curator on the assay. supported_by: - reference_id: PMID:23716655 supporting_text: the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. - term: id: GO:0140404 label: effector-mediated perturbation of host innate immune response by symbiont evidence_type: EXP original_reference_id: PMID:9437864 qualifier: involved_in review: summary: >- Thomas et al. cloned tomato Cf-4, which confers resistance through recognition of AVR4. The abstract does not describe the AVR4 assays, but cloning of Cf-4 would have required AVR4-dependent responses in transgenic plants; the curator read the full text. action: KEEP_AS_NON_CORE reason: >- Consistent with the well-established AVR4/Cf-4 gene-for-gene relationship; the annotation reflects host recognition (avirulence) rather than AVR4's intrinsic function and is redundant with the more specific GO:0080185. Full text not available here, so the curator's assay call is accepted. supported_by: - reference_id: PMID:9437864 supporting_text: In tomato, the Cf-4 and Cf-9 resistance genes map to the same location but confer resistance to Cladosporium fulvum through recognition of different avirulence determinants (AVR4 and AVR9) - term: id: GO:0140404 label: effector-mediated perturbation of host innate immune response by symbiont evidence_type: TAS original_reference_id: PMID:25902074 qualifier: involved_in review: summary: >- Iida et al. surveyed Japanese C. fulvum isolates and found Avr4 mutations (mostly cysteine substitutions and a frameshift) that allow strains to overcome Cf-4 resistance, and tested HR-inducing activity of wild-type and mutant Avr4 alleles by PVX expression. action: KEEP_AS_NON_CORE reason: >- The paper supports the avirulence/recognition role of AVR4 (HR in Cf-4 plants, lost in variant alleles). As with the other GO:0140404 rows this is a host recognition outcome and is redundant with GO:0080185; retained as non-core. supported_by: - reference_id: PMID:25902074 supporting_text: their encoded proteins trigger a hypersensitive response (HR) in host plants carrying the corresponding Cf-2, Cf-4, Cf-4E, Cf-5, and Cf-9 genes - term: id: GO:0140593 label: host apoplast evidence_type: IDA original_reference_id: PMID:12736265 qualifier: located_in review: summary: >- AVR4 is secreted into the tomato leaf apoplast during infection, where it is detected in apoplastic fluid; virulent isoforms are absent from apoplastic fluid because they are protease-sensitive. action: NEW reason: >- A more specific, symbiont-side location than extracellular region, directly supported by detection of AVR4 isoforms (or their absence) in tomato apoplastic fluid and by apoplastic expression studies. This is a location term, so the participation test does not apply. supported_by: - reference_id: PMID:12736265 supporting_text: resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid - reference_id: PMID:9090881 supporting_text: Polyclonal antibodies raised against the AVR4 elicitor, however, did not detect AVR4 isoforms in MM-Cf4 plants infected by the different virulent strains, indicating that these isoforms are unstable. - term: id: GO:0009277 label: fungal-type cell wall evidence_type: IDA original_reference_id: PMID:17153926 qualifier: located_in review: summary: >- In situ fluorescence showed that AVR4 binds to the cell walls of C. fulvum hyphae during tomato infection; this is where it exerts its protective function. action: NEW reason: >- Direct in situ localisation of the protein on the fungus's own cell wall during infection; captures the operative site of the core function. supported_by: - reference_id: PMID:17153926 supporting_text: In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato core_functions: - description: >- Secreted non-enzymatic chitin-binding lectin (CBM14) that coats chitin in the cell walls of the fungus's own hyphae during apoplastic colonisation of tomato, shielding the wall from hydrolysis by plant chitinases and thereby allowing the fungus to withstand this host antifungal defence. molecular_function: id: GO:0008061 label: chitin binding directly_involved_in: - id: GO:0141177 label: symbiont-mediated evasion of recognition by host innate immune effector locations: - id: GO:0009277 label: fungal-type cell wall - id: GO:0140593 label: host apoplast supported_by: - reference_id: PMID:30148881 supporting_text: Collectively, results from the protection assays show that mutations in the ChBD of CfAvr4 that decrease or abolish its affinity for (GlcNAc)6 also reduce the protein’s ability to protect fungal germlings against chitinases and thus perform its biological function. - reference_id: PMID:17153926 supporting_text: In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. - reference_id: PMID:14769793 supporting_text: By this mechanism AVR4 is likely to effectively shield chitin on the fungal cell wall, preventing the cell wall from being degraded by plant chitinases. - reference_id: PMID:17849712 supporting_text: This is the first report on the intrinsic function of a fungal avirulence protein that has a counter-defensive activity required for full virulence of the pathogen. - reference_id: file:FULFL/AVR4/AVR4-deep-research-falcon.md supporting_text: It coats accessible chitin in fungal cell walls, limiting hydrolysis by plant chitinases during infection. references: - id: file:FULFL/AVR4/AVR4-record-evidence.yaml title: Compact archived record and sequence evidence for Q00363 - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: PMID:10998185 title: Specific HR-associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and non-host plants. findings: [] - id: PMID:16167771 title: The Cf-4 and Cf-9 resistance genes against Cladosporium fulvum are conserved in wild tomato species. findings: [] - id: PMID:23716655 title: Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. findings: [] - id: PMID:25902074 title: Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum. findings: [] - id: PMID:30148881 title: Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein. findings: - statement: Crystal structure of AVR4 with (GlcNAc)6; W100A and D102A abolish binding and fail to protect hyphae from chitinases; chitin-binding mutants that remain protease-stable are still recognised by Cf-4. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text cached and read. Supports chitin binding and chitinase protection; does not test PAMP sequestration (decoy activity). - id: PMID:9437864 title: Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies sequences that determine recognitional specificity in Cf-4 and Cf-9. findings: [] - id: PMID:17849712 title: The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor. findings: - statement: Silencing of Avr4 in C. fulvum reduces virulence on tomato; heterologous Avr4 expression increases virulence of fungi with exposed wall chitin. reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1094/MPMI-20-9-1092); abstract only cached. - id: PMID:17153926 title: Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection. findings: - statement: Avr4 binds chitin specifically, protects fungi against plant chitinases, and binds C. fulvum cell walls in situ during infection. reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1094/MPMI-19-1420); abstract only cached. - id: PMID:14769793 title: 'Binding of the AVR4 elicitor of Cladosporium fulvum to chitotriose units is facilitated by positive allosteric protein-protein interactions: the chitin-binding site of AVR4 represents a novel binding site on the folding scaffold shared between the invertebrate and the plant chitin-binding domain.' findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1074/jbc.M312594200); thermodynamics of chito-oligomer binding and cooperative binding model. - id: PMID:12736265 title: Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis, circumvent Cf-4-mediated resistance, but retain their chitin binding ability. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1074/jbc.M212196200). - id: PMID:27401545 title: Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1105/tpc.15.00893). Structure is of the P. fuligena ortholog Pf-Avr4; Cf-Avr4 included in comparative binding and chitinase-protection assays. - id: PMID:9090881 title: The biotrophic fungus Cladosporium fulvum circumvents Cf-4-mediated resistance by producing unstable AVR4 elicitors. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified (DOI 10.1105/tpc.9.3.367). - id: file:FULFL/AVR4/AVR4-deep-research-falcon.md title: Deep research report on AVR4 (Falcon) findings: [] suggested_questions: - question: >- Does AVR4 bound to hyphal walls reduce the release of chitin oligomers that would otherwise be perceived by tomato chitin receptors (e.g. CEBiP/LYK homologues), i.e. does wall shielding also contribute to evasion of chitin recognition (GO:0141141) in addition to protection from chitinases? - question: >- GO:0080185 (effector-mediated activation of plant hypersensitive response) is currently an is_a descendant of GO:0140403 (effector-mediated suppression of host innate immune response). Is this placement intended, given that activation of HR is the opposite outcome to suppression? suggested_experiments: - description: >- Test whether purified AVR4 (wild type versus non-binding W100A/D102A) affects chitin-oligomer-induced ROS burst or medium alkalinisation in tomato cell cultures, both with soluble (GlcNAc)6/8 and with intact hyphal walls challenged by tomato chitinases, to separate sequestration from wall shielding. - description: >- Generate a clean Avr4 deletion in C. fulvum (rather than silencing) and complement with chitin-binding-deficient alleles to quantify the contribution of chitin binding to virulence on Cf-0 tomato.