PIT2 (UMAG_01375; "protein important for tumours 2") encodes a small (120-residue) secreted effector of the maize smut fungus Ustilago maydis (Mycosarcoma maydis). It is encoded in the pit cluster next to the transmembrane protein Pit1, shares a bidirectional promoter with pit1, and is strongly induced during biotrophic growth, partly through direct binding of the unfolded-protein-response regulator Cib1 to the pit1/pit2 promoter. After signal-peptide cleavage, Pit2 is secreted to the biotrophic interface and spreads through the apoplast around colonized maize cells. There it binds and inhibits a set of apoplastic maize papain-like cysteine proteases (CP1A, CP1B, CP2 and XCP2, but not cathepsin B) whose activity is linked to salicylic-acid-associated defence and to release of the immune signalling peptide Zip1 from PROZIP1. Inhibition depends on a conserved 14-residue motif (PID14, residues 44-57); mutation of its central aromatic residues abolishes inhibition and virulence without affecting secretion, and a synthetic PID14 peptide inhibits the proteases on its own. Pit2 behaves as a substrate mimic: host proteases cleave it, releasing an inhibitory core peptide from PID14 that then blocks them. Related motifs occur in other plant-associated fungi and bacteria. pit2 deletion mutants penetrate maize but trigger defence responses, stop proliferating and fail to induce tumours.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | EXP PMID:21692877 Two linked genes encoding a secreted effector and a membrane... | ACCEPT | Summary: Doehlemann et al. (2011) showed by fluorescent tagging that Pit2 is secreted to the biotrophic interface during maize colonization (UniProt: Secreted, ECO:0000269 from this paper). Reason: Pit2 has a signal peptide and was observed outside fungal hyphae in planta. Extracellular region is correct. Only the abstract is cached, but it states the secretion directly. The more specific host-side location (host apoplast) is added as a NEW annotation below. Supporting Evidence: PMID:21692877 Pit1 localizes to the fungal plasma membrane at hyphal tips, endosomes and vacuoles while Pit2 is secreted to the biotrophic interface. PMID:23459172 Pit2 is secreted to the biotrophic interface and spreads in the apoplastic space around colonized maize cells. |
| GO:0005576 extracellular region | EXP PMID:27093436 Unfolded Protein Response (UPR) Regulator Cib1 Controls Expr... | ACCEPT | Summary: Hampel et al. (2016) detected Pit2-mCherry in axenic culture supernatants, with secretion increased by ER stress in a Cib1-dependent manner. Reason: Direct detection of the secreted protein in culture supernatant supports extracellular region. The main subject of the paper is regulation of pit2 expression and secretion by the UPR, which is fungal-side regulation and not a function of Pit2 itself. Supporting Evidence: PMID:27093436 Induction of DTT-mediated ER stress resulted in strongly increased levels of secreted Pit2-mCherry in the supernatant, compared to the untreated WT. PMID:27093436 Secretion of Pit2-mCherry is strongly increased upon ER stress and dependent on Cib1 |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the UniProt subcellular location "Secreted", which is itself experimentally supported. Reason: Correct mapping, consistent with the two experimental annotations. Supporting Evidence: PMID:23459172 Pit2 is secreted to the biotrophic interface and spreads in the apoplastic space around colonized maize cells. |
| GO:0004869 cysteine-type endopeptidase inhibitor activity | IDA PMID:23459172 Compatibility in the Ustilago maydis-maize interaction requi... | NEW | Summary: Recombinant Pit2 inhibits maize apoplastic cysteine protease activity in apoplastic fluid fractions in a concentration-dependent way, and inhibits individually expressed CP1A, CP2 and XCP2 but not CatB. It binds CP1A, CP1B, CP2 and XCP2 in yeast two-hybrid assays and co-immunoprecipitates with CP2. A PID14-motif mutant (Pit2mut49-53) loses inhibition, and the synthetic PID14 peptide inhibits on its own. Reason: The core molecular function of Pit2 is absent from GOA, which carries only a location term. Direct in vitro assays with purified protein, target specificity, and loss-of-function mutants show that Pit2 itself has this activity, so the participation test is met. Comparator check: the Phytophthora infestans apoplastic PLCP-inhibitor effector EPIC2B (D0NBV3) carries GO:0004869 and GO:0030414 in GOA, so the term is the one GO uses for effectors in this role. GO:0004869 is the specific child of peptidase inhibitor activity and matches the papain-like (C1A) target class. The 2019 substrate-mimic model (processing releases the inhibitory core) does not change the activity term, because inhibition is carried out by the Pit2 polypeptide or its processed product. Supporting Evidence: PMID:23459172 Together, we conclude from these data that Pit2 functions as an inhibitor of the apoplastic maize cysteine proteases CP1A, CP2 and XCP2 but not CatB. PMID:23459172 By contrast to the native protein, Pit2mut49β53 did not cause a significant inhibition of protease activity when being applied at similar concentrations (Figure 6A). PMID:30952847 Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. |
| GO:0140403 effector-mediated suppression of host innate immune response | IMP PMID:23459172 Compatibility in the Ustilago maydis-maize interaction requi... | NEW | Summary: Pit2 suppresses salicylic-acid-associated apoplastic immunity in maize by inhibiting the host papain-like cysteine proteases that drive it. pit2 deletion mutants trigger broad defence responses and host-cell collapse soon after penetration. Inhibitor-motif mutants that are still secreted and localize correctly do not restore virulence, which ties the virulence function to protease inhibition. Reason: This symbiont-side process term describes what the effector does to the host. It is chosen over plant-side defence terms or a bare virulence phenotype. Participation test: Pit2 itself performs the step, because it inhibits the host immune proteases directly. This is not just necessity evidence: the PID14 point mutants separate protease inhibition from secretion and localization, and the free PID14 peptide restores virulence partially. Comparator check: EPIC2B, which has the same role, carries the parent term GO:0052170 (symbiont-mediated suppression of host innate immune response). GO:0140403 is the effector-specific child and fits a secreted protein. The more specific GO:0140502 (suppression of host salicylic acid-mediated innate immune signaling) was not used. The target proteases are activated by SA and process PROZIP1 into Zip1, which amplifies SA signalling, but Pit2 does not act on an SA signalling component itself (see suggested_questions). Supporting Evidence: PMID:23459172 We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. PMID:23459172 Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. PMID:30952847 Both features, substrate recognition by PLCPs and subsequently PLCP inhibition, are required for full Pit2 virulence function in maize file:MYCMD/PIT2/PIT2-deep-research-falcon.md Maize CP1 and CP2, but not XCP2 or CatB, processed PROZIP1 to release immune-active Zip1 |
| GO:0140593 host apoplast | IDA PMID:23459172 Compatibility in the Ustilago maydis-maize interaction requi... | NEW | Summary: Pit2-mCherry accumulates at the biotrophic interface around intercellular hyphae and spreads through the apoplastic space around colonized maize cells, which is where its protease targets act. Reason: More specific than extracellular region and describes the site of action (the host apoplast that contains the target PLCPs). This matches the location used for the companion apoplastic effector CMU1 before it is taken up into host cells. Supporting Evidence: PMID:23459172 Pit2 is secreted to the biotrophic interface and spreads in the apoplastic space around colonized maize cells. PMID:23459172 Both the mCherry-tagged Pit2Ξ44β57 and Pit2mut49β53 proteins were found to accumulate in the biotrophic interface, surrounding the intercellular hyphae. |
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Download this section (compressed HTML)Q: Pit2 targets the maize proteases CP1 and CP2, which process PROZIP1 into the Zip1 peptide that amplifies salicylic acid signalling. Is this enough to annotate Pit2 to GO:0140502 (effector-mediated suppression of host salicylic acid-mediated innate immune signaling), as for CMU1? Or should that term be kept for effectors that act on SA biosynthesis or signalling components directly?
Q: Under the substrate-mimic model, is the active inhibitor the intact protein, the processed PID14-derived core peptide, or both? If only the processed peptide inhibits in planta, should the activity be recorded for a processed chain feature rather than the precursor?
Q: A 2025 preprint (not peer reviewed) proposes that a Pit2-derived peptide (WWFGFTGSL) activates the fungal GPCR Gpe1 and promotes fungal proliferation. If confirmed, this would be a second, fungus-side signalling role (receptor ligand activity) and would need separate annotation.
Experiment: Measure Zip1 release and free SA in maize leaves infected with wild-type, pit2 deletion and PID14-mutant strains, to test directly whether Pit2 suppresses the PROZIP1-Zip1-SA amplification loop in planta.
Experiment: Determine a co-crystal or cryo-EM structure of CP2 or XCP2 with the processed Pit2 inhibitory peptide, to define the binding mode and to tell apart substrate-like and inhibitor-like interactions at the active site.
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