ACE1 (Avirulence Conferring Enzyme 1) of the rice blast fungus Pyricularia (Magnaporthe) oryzae is a large (about 4,035 aa) hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) with the domain architecture KS-AT-DH-CMeT-ER0-KR-ACP-C-A-T-R. Its iterative highly reducing PKS module builds a C-methylated polyketide chain from acetate units (the embedded enoyl reductase domain is inactive, and enoyl reduction is supplied in trans by the partner enzyme RAP1), and its single NRPS module condenses the polyketide with tyrosine, after which the chain is probably released by the terminal reductase domain. ACE1 is the first enzyme of a 15-gene, infection-specific secondary metabolite cluster that is predicted to produce a tyrosine-derived cytochalasan; the native end product has not been identified. ACE1 is expressed only in appressoria at the onset of host penetration and is retained in the appressorial cytoplasm rather than secreted. Rice cultivars carrying the resistance gene Pi33 recognize isolates carrying a catalytically active ACE1, so the avirulence signal is thought to be the metabolite made by the ACE1 pathway rather than the ACE1 protein itself; a KS active-site Cys-to-Ala mutant loses avirulence.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the NRPS condensation (C) domain (IPR001242). The term is correct but uninformative. ACE1 carries a complete NRPS module (C-A-T) that attaches tyrosine to the polyketide chain built by the PKS module, which is the activity the condensation domain contributes to. Reason: The root catalytic activity term carries no information. The C domain belongs to the single NRPS module of ACE1, and heterologous co-expression of ACE1 with RAP1 gave a tyrosine-containing amide, showing that the NRPS module condenses an amino acid onto the polyketide (PMID:29142718). Fungal PKS-NRPS hybrids with experimental data (e.g. iccA, iliA) are annotated to non-ribosomal peptide synthetase activity alongside polyketide synthase activity, so the same specific term is proposed here. Proposed replacements: non-ribosomal peptide synthetase activity Supporting Evidence: PMID:29142718 In this case it appears that tyrosine is used as the amino acid and attached to the polyketide in the same way as observed in the cases of pretenellin A 1 and preaspyridone 3. file:PYRO7/ACE1/ACE1-uniprot.txt ACE1 has the following architecture: KS-AT-DH-MT-KR-PCP-C-A-T-R. |
| GO:0004312 fatty acid synthase activity | IEA GO_REF:0000118 | MODIFY | Summary: TreeGrafter transfer from a PANTHER node of the type I PKS/FAS superfamily. ACE1 shares the KS-AT-DH-KR-ACP chain-building domains with fatty acid synthases, but it is a PKS-NRPS whose PKS module makes a C-methylated polyketide (a heptaketide pyrone when expressed alone, a nonaketide fused to tyrosine with RAP1), not a long-chain fatty acid. Reason: The fatty acid synthase reaction (acetyl-CoA + n malonyl-CoA to a fully reduced long-chain fatty acid) is not what ACE1 catalyses. Its products are partially reduced, methylated polyketides, and the enzyme lacks a functional enoyl reductase (PMID:29142718). The shared fold is captured correctly by polyketide synthase activity, which is used for experimentally characterized fungal PKS and PKS-NRPS enzymes (e.g. lovB, iliA). Proposed replacements: polyketide synthase activity Supporting Evidence: PMID:29142718 CC values showed intact acetate incorporations consistent with the heptaketide origin of 6. PMID:29142718 The biosynthesis of 6 and 7 do not require an enoyl reductase step, consistent with the observation that the ACE1 ER domain is predicted to be inactive. file:PYRO7/ACE1/ACE1-deep-research-falcon.md The embedded ER⁰ is predicted to be catalytically inactive |
| GO:0004315 3-oxoacyl-[acyl-carrier-protein] synthase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the beta-ketoacyl synthase active-site signature (IPR018201). The ACE1 KS domain performs the decarboxylative Claisen condensation of an acyl chain with malonyl bound to its own carrier domain, which matches the reaction in this term. Mutating the conserved KS active-site cysteine (C183A) abolished avirulence without affecting expression or localization. Reason: The KS partial activity is real and is supported by the C183A mutant (PMID:15319478), but it is one step of the overall polyketide synthase activity, which is the better description of what ACE1 does. Kept as a non-core, domain-level activity. Supporting Evidence: PMID:15319478 a universally conserved Cys residue of the KS active site is essential for its catalytic activity PMID:15319478 These results demonstrate that the ace1 C183A : GFP allele is unable to confer avirulence. |
| GO:0005737 cytoplasm | EXP PMID:15319478 A putative polyketide synthase/peptide synthetase from Magna... | ACCEPT | Summary: A functional Ace1-GFP fusion, expressed from the native promoter, was seen only in the cytoplasm of appressoria during penetration of rice and barley leaves. It stayed in the appressorium after infection hyphae emerged and was never detected outside it. ACE1 has no signal peptide. Reason: Direct localization of a functional fusion protein (it conferred avirulence) in the native context. A cytoplasmic location fits a PKS-NRPS biosynthetic enzyme, and it is the evidence that the protein is not secreted. Supporting Evidence: PMID:15319478 Fluorescence of the Ace1-GFP protein was detected exclusively in the cytoplasm of appressoria PMID:15319478 Ace1 does not display a signal peptide sequence at its N terminus and was predicted to be cytoplasmic by PSORT II software |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping, derived from the same Ace1-GFP experiment that supports the EXP row. Reason: Consistent with the experimental localization (PMID:15319478). It duplicates the EXP annotation but is correct. Supporting Evidence: PMID:15319478 Fluorescence of the Ace1-GFP protein was detected exclusively in the cytoplasm of appressoria |
| GO:0006633 fatty acid biosynthetic process | IEA GO_REF:0000120 | MODIFY | Summary: Combined IEA from the KS active-site signature and the PKS/FAS PANTHER node. There is no evidence that ACE1 makes fatty acids. Its PKS module makes polyketide chains that are condensed with tyrosine, as the first step of a predicted cytochalasan pathway. Reason: The KS signature is shared by fatty acid synthases and polyketide synthases and does not tell them apart. Isotope labelling and structure determination of the ACE1 products show polyketide biosynthesis (PMID:29142718), so the process should be polyketide biosynthetic process. Proposed replacements: polyketide biosynthetic process Supporting Evidence: PMID:29142718 CC values showed intact acetate incorporations consistent with the heptaketide origin of 6. PMID:29142718 It appears likely, therefore, that ACE1 encodes a biosynthetic protein which makes a small molecule consisting of a polyketide fused to an amino acid. |
| GO:0016740 transferase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the acyl transferase domain superfamily (IPR001227), corresponding to the AT domain that loads malonyl extender units onto the carrier domain. Correct but far too general. Reason: The AT domain is one part of the polyketide synthase module, and transferase activity describes none of what ACE1 does. The informative replacement is polyketide synthase activity, the overall activity of the PKS module that contains the AT domain (PMID:29142718). Proposed replacements: polyketide synthase activity Supporting Evidence: PMID:29142718 The 12.4 kb ACE1 gene encodes an enzyme consisting of a fungal highly-reducing polyketide synthase |
| GO:0016746 acyltransferase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the thiolase-like fold of the KS domain. KS condensation and AT loading are both acyl transfers, so the term is correct (UniProt gives EC 2.3.1.- for ACE1), but it is general. Reason: Correct as a general description of the KS and AT partial reactions. The core activity is better captured by polyketide synthase activity (PMID:29142718). Supporting Evidence: PMID:29142718 The 12.4 kb ACE1 gene encodes an enzyme consisting of a fungal highly-reducing polyketide synthase PMID:15319478 a universally conserved Cys residue of the KS active site is essential for its catalytic activity |
| GO:0031177 phosphopantetheine binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the phosphopantetheine attachment site found in carrier domains. ACE1 has two carrier domains, the PKS acyl carrier protein (ACP/PCP) and the NRPS thiolation (T) domain, each of which carries a 4'-phosphopantetheine prosthetic group to which the growing chain is tethered. Reason: Correct from the domain architecture. Binding of the covalent prosthetic group is a feature of the carrier domains rather than the biological function of ACE1, so it is kept as non-core. Supporting Evidence: file:PYRO7/ACE1/ACE1-uniprot.txt ACE1 has the following architecture: KS-AT-DH-MT-KR-PCP-C-A-T-R. |
| GO:0044550 secondary metabolite biosynthetic process | IEA GO_REF:0000118 | ACCEPT | Summary: TreeGrafter annotation. ACE1 is the core synthase of an infection-specific secondary metabolite gene cluster. It makes polyketide and polyketide-tyrosine products when expressed heterologously, and the predicted end product of the pathway is a tyrosine-derived cytochalasan. Reason: Well supported by cluster analysis (PMID:18433432) and heterologous expression (PMID:29142718). Because the native product is unknown, this general term, together with polyketide biosynthetic process, is the appropriate level of specificity. Supporting Evidence: PMID:18433432 Analysis of the M. grisea genome revealed that ACE1 is located in a cluster of 15 genes, of which 14 are potentially involved in secondary metabolism PMID:29142718 the likely ultimate product of the ACE1 pathway will be a member of the tyrosine-derived cytochalasans |
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Download this section (compressed HTML)Q: Should ACE1 carry any host-interaction process term? The protein stays in the appressorial cytoplasm and is not recognized itself; the Pi33-recognized signal is an unidentified pathway metabolite. Existing symbiont-side terms such as effector-mediated activation of plant hypersensitive response by symbiont (GO:0080185) are framed around an effector acting on the host, so it is unclear whether a biosynthetic enzyme of an avirulence metabolite should be annotated to them, or whether such terms should go only to a gene product whose molecule is actually perceived.
Q: What is the native cytochalasan end product of the ACE1 cluster, and is it perceived directly by Pi33 (or a Pi33-linked receptor) in rice?
Experiment: Express the full ACE1 cluster (ACE1, RAP1, ORFZ, ORF3, CYP1-4, OXR1, OXR2) in a heterologous host, or engineer constitutive expression of the cluster-specific transcription factor in M. oryzae, to isolate the mature cytochalasan, then test whether it triggers a Pi33-dependent response on rice leaves.
Type: heterologous expression and metabolite bioassay
Experiment: In vitro adenylation assays (e.g. ATP-PPi exchange or hydroxamate assays) with the excised ACE1 A domain to define its amino acid specificity directly.
Type: in vitro biochemistry
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