AVR-Pita is a secreted effector of the rice blast fungus Pyricularia (Magnaporthe) oryzae and the founding member of a small gene family (AVR-Pita1/2/3) that sits in unstable, often subtelomeric genomic locations. The 224-residue precursor has an N-terminal signal peptide followed by a fungal aspzincin (peptidase M35, deuterolysin-like) domain with an intact HExxH zinc-binding motif, so the protein is predicted to be a neutral zinc metalloprotease; no catalytic activity, substrate or product has ever been demonstrated for it, and the gene is dispensable for pathogenicity. During infection the protein is secreted through the Golgi-independent route into the biotrophic interfacial complex and delivered into the cytoplasm of invaded rice cells, where it localises to host mitochondria, binds the cytochrome c oxidase assembly protein OsCOX11 and raises COX activity; this lowers the reactive oxygen species burst triggered by the fungal pattern chitin and increases susceptibility, making AVR-Pita a suppressor of pattern-triggered immunity. In rice lines with the matching resistance specificity the same protein is detected and triggers an R-gene-dependent hypersensitive response and race-specific resistance; recognition was long attributed to the NLR Pi-ta but has since been shown to depend instead on the atypical armadillo-repeat membrane protein Ptr. Mutations in the predicted protease motif, frameshifts, transposon insertions and outright loss of the locus allow field isolates to escape this recognition, and the gene is correspondingly highly polymorphic and mobile in pathogen populations.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004222 metalloendopeptidase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from IPR029463 (lysine-specific metallo-endopeptidase domain). AVR-Pita does carry an intact aspzincin/M35 domain, and the original cloning paper inferred a neutral zinc metalloprotease from sequence similarity, but it also stated plainly that no catalytic activity had been shown, and none has been demonstrated since. The one study of what AVR-Pita does to a host protein found binding to, and functional modulation of, OsCOX11, not cleavage of it (PMID:33443630). Reason: This is the more specific of two overlapping domain-derived molecular function rows, and it adds two unverified layers to an already unverified catalytic claim: that the (undemonstrated) peptidase is an endopeptidase, and, through the family name of IPR029463, that it is lysine-specific. For this protein no substrate, cleavage site or kinetic measurement exists; the domain-level inference is kept on the GO:0008237 row instead. The mapping is not biologically absurd, since characterised M35 deuterolysins are endopeptidases, which is why this is marked as over-annotation rather than removed. Supporting Evidence: PMID:11090206 Although protease activity by this protein has not been demonstrated biochemically, it is implicated. PMID:11090206 The fungal metalloproteases showing the greatest sequence identity to AVR-Pita fall into families M35 and M36 in this superfamily of zinc-dependent metalloproteases. file:PYROR/AVR-Pita/AVR-Pita-deep-research-falcon.md Its classification as an M35 zinc metalloprotease is a *sequence/domain inference*, not a demonstrated catalytic reaction. PMID:15319478 AVR-Pita from the rice ( Oryza sativa ) pathogen Magnaporthe grisea encodes a putative zinc-dependent metalloprotease that is dispensable for pathogenicity |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping from the UniProt "Secreted" subcellular location. AVR-Pita has a predicted N-terminal signal peptide (residues 1-17 of F7J183) and behaves as a secreted protein in live-cell imaging during rice infection, accumulating in the biotrophic interfacial complex outside the fungal cell before delivery into the host cytoplasm. Reason: The secreted stage is real and experimentally visualised: AVR-Pita is one of the effectors shown to traffic through the brefeldin-A-insensitive, BIC-associated secretion system, and it is routinely classed as a cytoplasmic (host-translocated) effector, which requires passage out of the fungal cell. The term is broad but correct; the host-side compartment where the protein acts is captured separately by the new GO:0033650 annotation. Supporting Evidence: PMID:23774898 we tested additional cytoplasmic effectors AVR-Pita (ref. 8) (Fig. 4c), Bas1 (ref. 25) (Fig. 4c) and Bas107 PMID:38722963 The cytoplasmic effectors include known AVR effectors, such as Pwl2, Avr-Pita, and AvrPiz-t PMID:33443630 Avr-Pita is a putative neutral zinc metalloprotease with a signal peptide at its N-terminus |
| GO:0008237 metallopeptidase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the metallopeptidase catalytic domain superfamily (IPR024079). The F7J183 sequence does contain an intact aspzincin HExxH zinc-binding motif (HEMLH beginning at residue 177 of the 224-residue precursor, followed by the NEWEDYGY region), UniProt assigns a Zn(2+) cofactor and places the protein in the peptidase M35 family, and substituting the motif glutamate (E177D in the 223-residue numbering of the original papers) abolishes effector function. Reason: The family-level inference is defensible: the fold and the catalytic motif are present and unmutated in this allele. But it has stayed an inference for 25 years - no protease assay, substrate or product has been reported - the gene is dispensable for pathogenicity, and the original authors explicitly left open that the motif mutations act on recognition rather than on catalysis. The function for which AVR-Pita has evidence is binding and modulating the host protein OsCOX11 at host mitochondria to suppress chitin-triggered ROS, so the predicted peptidase activity is kept as a plausible non-core family attribute rather than asserted as a core function. Supporting Evidence: PMID:11090206 AVR-Pita appears to encode a neutral zinc metalloprotease, given its amino acid sequence similarity to fungal metalloproteases PMID:11090206 However, we do not rule out an alternative explanation for the effect of these missense mutations on AVR-Pita avirulence function PMID:10921881 The avr-pita protein with the putative active site glutamic acid replaced with aspartic acid (E177D) failed to confer avirulence in whole-plant infection assays file:PYROR/AVR-Pita/AVR-Pita-uniprot.txt Belongs to the peptidase M35 family. |
| GO:0033650 host cell mitochondrion | IDA PMID:33443630 The Fungal Effector Avr-Pita Suppresses Innate Immunity by I... | NEW | Summary: Han et al. (2021) expressed Avr-Pita:YFP in rice protoplasts and onion epidermal cells and found complete co-localisation with a mitochondrial marker protein and with MitoTracker, and co-localisation with OsCOX11:mCherry in mitochondria. The host mitochondrion is therefore the plant compartment in which the delivered effector is found and in which it engages its host target. Reason: GOA records only the fungal-side secreted location (GO:0005576); the compartment in which the translocated effector acts is not annotated. This is a cellular component term supported by direct imaging in plant cells, so it raises none of the participation concerns that apply to new process terms. GO:0030430 host cell cytoplasm was considered and deliberately not proposed: AVR-Pita is classed as a cytoplasmic effector, but the direct localisation evidence in rice cells is mitochondrial and the host-cell compartment is already stated by this term. Supporting Evidence: PMID:33443630 Avr-Pita:YFP perfectly co-localized with the mitochondrial marker Rflb:mCherry (Wang et al., 2006) in rice protoplasts and with the mitochondrial dye MitoTracker in onion epidermal cells PMID:33443630 These results indicate that the fungal effector Avr-Pita targets the host mitochondria and binds OsCOX11 |
| GO:0052034 effector-mediated suppression of host pattern-triggered immunity | IDA PMID:33443630 The Fungal Effector Avr-Pita Suppresses Innate Immunity by I... | NEW | Summary: Ectopic expression of Avr-Pita in rice suppressed PAMP-triggered defence responses and enhanced susceptibility to M. oryzae. Mechanistically the effector binds the COX assembly protein OsCOX11 (yeast two-hybrid with the mature Avr-Pita49-224, confirmed by pull-down), raises cytochrome c oxidase activity, and lowers the reactive oxygen species burst elicited by the fungal pattern chitin. Reason: This is the only mechanistic virulence function established for AVR-Pita and it is absent from GOA, which holds only domain- and location-derived rows. The participation test is satisfied by the effector doing the work itself: AVR-Pita is the entity that binds OsCOX11 and the entity whose expression raises COX activity, and that raised COX activity is the step that damps the chitin-triggered ROS burst; the claim does not rest on a knockout phenotype showing mere necessity. GO:0052034 is the right level because the suppressed output measured is immunity triggered by a microbe-associated molecular pattern (chitin), not a salicylate-specific or effector-triggered branch. Comparator check: the symbiont-side effector terms are the established convention for curated fungal effectors in this repository rather than a term type that avoids substrates - for example AVR-Pia (genes/PYROR/AVR-Pia) carries the sibling term GO:0080185 - so this is a genuine gap in the IEA-only GOA record for AVR-Pita. Supporting Evidence: PMID:33443630 ectopically expressing Avr-Pita in rice enhances susceptibility to M. oryzae and suppresses pathogen-associated molecular pattern (PAMP)-triggered defense responses PMID:33443630 ROS accumulation was significantly suppressed in PUbi::Avr-Pita and PUbi::OsCOX11 plants, whereas the ROS burst was strongly delayed in oscox11/OsCOX11 cells after chitin treatment PMID:33443630 COX activity was much higher in PUbi::Avr-Pita and PUbi::OsCOX11 plants than in the WT both before and after chitin treatment |
| GO:0080185 effector-mediated activation of plant hypersensitive response by symbiont | IDA PMID:10921881 Direct interaction of resistance gene and avirulence gene pr... | NEW | Summary: AVR-Pita is the ligand whose detection in rice carrying the matching resistance specificity triggers an R-gene-dependent hypersensitive response and race-specific resistance. Introducing the cloned gene into virulent isolates confers avirulence on those rice cultivars (PMID:11090206), and transient expression of AVR-Pita176 inside rice cells elicits the resistance response, which is lost in the same protease-motif mutants that lose avirulence in whole-plant infection (PMID:10921881). Reason: Participation test: the effector itself performs the recognised step - it is the elicitor, and effector-side point mutations abolish the host response, so the claim is not simply that the gene is required for an outcome produced by host proteins. GO:0080185 is defined for exactly this symbiont-side role, including indirect recognition through R proteins, which matters here because the receptor assignment has changed: recognition was attributed to the NLR Pi-ta on the basis of direct AVR-Pita176-Pi-ta LRD binding, but RNAi and CRISPR genetics later showed it depends solely on the atypical protein Ptr (PMID:38834685). That reassignment changes which host protein detects the effector, not the fact that the effector triggers the hypersensitive response, so the symbiont-side term stands. Comparator check: AVR-Pia and the Fulvia fulva effector AVR9 reviewed in this repository carry the same term for the same role, and GOA has no process annotation at all for AVR-Pita. Supporting Evidence: PMID:11090206 When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. PMID:10921881 two separate mutations in the AVR-Pita protease motif that eliminate avirulence in fungal infection assays also eliminate elicitor activity in the transient expression assay PMID:10921881 These data suggest that the AVR-Pita 176 protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta -mediated defense response PMID:38834685 we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it |
Loading supporting content…
Download this section (compressed HTML)Q: Is AVR-Pita a catalytically active metalloprotease at all, and if so what does it cleave in the rice cell? The HExxH motif is intact and motif mutations abolish effector function, but no cleavage has ever been measured, and the characterised host interaction with OsCOX11 was shown as binding.
Suggested experts: Barbara Valent
Q: Does AVR-Pita cleave OsCOX11, or does it act as a non-catalytic activator of COX assembly? Distinguishing these decides whether a molecular function term can be asserted at all and, if so, whether has_input OsCOX11 is appropriate.
Suggested experts: Letian Chen
Q: How should GO handle avirulence effectors whose recognising receptor has been reassigned, as here from the NLR Pi-ta to Ptr? The symbiont-side term GO:0080185 is unaffected, but host-side annotations made under the Pi-ta model may need re-curation onto Ptr.
Experiment: Express and refold the mature AVR-Pita aspzincin domain (residues about 49-224) and assay zinc-dependent peptidase activity against generic substrates (azocasein, FRETS peptide libraries) and against recombinant OsCOX11, comparing wild type with the HExxH glutamate mutant. This is the missing experiment behind every current molecular function annotation on this protein.
Experiment: Test whether catalysis is required for virulence as opposed to recognition: complement an AVR-Pita deletion isolate with wild-type and catalytic-glutamate mutant alleles and score chitin-triggered ROS suppression and COX activity in planta on rice lacking the recognising Ptr allele, so that the avirulence phenotype does not mask the virulence readout.
Experiment: Map the AVR-Pita import route to host mitochondria - whether delivery into the rice cytoplasm via the biotrophic interfacial complex is followed by canonical TOM/TIM import or by association with the outer membrane - using submitochondrial fractionation and protease-protection assays on infected tissue rather than on ectopically expressed protein.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)