PWL2 (Pathogenicity toward Weeping Lovegrass 2; MGG_04301, present in two copies in strain 70-15) encodes a small (145 aa), glycine-rich, signal-peptide-bearing cytoplasmic effector of the rice blast fungus Magnaporthe oryzae (Pyricularia oryzae). Its expression is induced during biotrophic invasion of living rice cells and is controlled by the Pmk1 MAP kinase. Pwl2 is secreted by invasive hyphae through a brefeldin A-insensitive, exocyst- and Sso1 t-SNARE-dependent route into the biotrophic interfacial complex (BIC), a plant membrane-rich structure at the hyphal tip, from where it is translocated into the rice cytoplasm, apparently by host clathrin-mediated endocytosis, and moves into neighbouring uninvaded cells. The mature protein adopts a MAX (Magnaporthe Avrs and ToxB-like) beta-sandwich fold with a C-terminal helical extension and binds the heavy-metal-associated (HMA) domain of host small HMA proteins of the HIPP43 type with nanomolar affinity through a large multi-part interface. Binding displaces HIPP43 from plasmodesmata into cytoplasmic mobile bodies; in planta expression of Pwl2 attenuates chitin- and flg22-induced reactive oxygen bursts and increases blast susceptibility, while deletion of the PWL2 copies reduces disease severity on susceptible hosts. In hosts carrying a cognate immune receptor the same protein acts as an avirulence and host-range determinant: it prevents infection of weeping lovegrass (Eragrostis curvula) and is recognised by the barley NLR MLA3. Natural alleles such as pwl2-2 escape this recognition while retaining HIPP43 binding, and a rice Pik NLR engineered to carry the OsHIPP43 HMA domain in place of its integrated HMA domain recognises Pwl2 and related PWL effectors.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | EXP PMID:20435900 Translocation of Magnaporthe oryzae effectors into rice cell... | ACCEPT | Summary: Live-cell imaging of fluorescent PWL2 secreted by invasive hyphae showed it accumulating in the biotrophic interfacial complex (BIC), outside the fungal cell, before translocation into rice cells (Khang et al. 2010). Reason: Pwl2 is a signal-peptide-bearing secreted effector whose first post-secretion location is the BIC, an extra-fungal, host-derived compartment. GO has no BIC term, so extracellular region is the accurate if general location for the secreted, pre-translocation pool. Supporting Evidence: PMID:20435900 continued to accumulate in BICs after IH were growing elsewhere PMID:23774898 fluorescent cytoplasmic effectors accumulate at BICs following photobleaching, and this process is BFA insensitive |
| GO:0005576 extracellular region | EXP PMID:26472068 Live-cell imaging of rice cytological changes reveals the im... | ACCEPT | Summary: Pwl2-mCherry secreted by invasive hyphae accumulates in BICs as puncta during rice infection (Mochizuki et al. 2015). Reason: Direct live-cell imaging of the secreted effector at the extra-fungal BIC supports the location. Supporting Evidence: PMID:26472068 These observations also reveal that Pwl2:mCherry is localized in BICs as puncta. |
| GO:0005576 extracellular region | EXP PMID:30182232 Visualizing the Movement of Magnaporthe oryzae Effector Prot... | ACCEPT | Summary: Methods chapter describing live-cell imaging of fluorescent PWL2 delivered by invasive hyphae into rice cells (Jones and Khang 2018); only the abstract is cached. Reason: Abstract-only cache, but the chapter uses fluorescent PWL2 as the reference translocated effector and the secreted BIC pool is consistent with the primary studies, so the curator's annotation is retained. Supporting Evidence: PMID:30182232 express the fluorescent protein-tagged effector known as PWL2 |
| GO:0005576 extracellular region | EXP PMID:31749824 Green Fluorescent Protein- and Discosoma sp. Red Fluorescent... | ACCEPT | Summary: Rice organelle-marker resource paper in which a fungal strain expressing PWL2-mCherry fused to a nuclear localization signal was used to demonstrate effector entry into rice cells (Chen et al. 2019). Reason: This paper's evidence chiefly supports host-cell translocation; the extracellular pool is implied by secretion from the fungus. Consistent with the primary data, so retained, although it is a weak independent source for the extracellular location specifically. Supporting Evidence: PMID:31749824 oryzae isolate carrying a PWL2:mCherry : NLS expression cassette |
| GO:0005576 extracellular region | EXP PMID:34236677 Characterizing the Secretion Systems of Magnaporthe oryzae. | ACCEPT | Summary: Protocol showing that secretion of Pwl2 from invasive hyphae is brefeldin A-insensitive and SNARE- and exocyst-dependent, unlike the apoplastic effector Bas4 (Oliveira-Garcia and Valent 2021). Reason: Supports secretion of Pwl2 out of the fungal cell, by a nonconventional route. Supporting Evidence: PMID:34236677 secretion of Pwl2 is BFA insensitive and follows a nonconventional secretion pathway that is Snare and Exocyst dependent |
| GO:0005576 extracellular region | EXP PMID:38653755 Phytoalexin sakuranetin attenuates endocytosis and enhances ... | ACCEPT | Summary: Study of the rice phytoalexin sakuranetin using rice lines expressing PWL2-GFP, showing that PWL2-GFP is membrane-associated and internalised by clathrin-mediated endocytosis, which sakuranetin attenuates (Jiang et al. 2024). Reason: The evidence comes from heterologous expression of PWL2 in rice rather than from fungal secretion, but it is consistent with Pwl2 reaching the host cell surface from outside and then being endocytosed. Supporting Evidence: PMID:38653755 demonstrating that PWL2 is also internalized by CME |
| GO:0005576 extracellular region | EXP PMID:38722963 A pharmacological approach to investigating effector translo... | ACCEPT | Summary: Clathrin heavy chain inhibitors (ES9, ES9-17) alter Pwl2-mRFP localization within BICs during infection, consistent with secreted Pwl2 being taken up by host endocytosis (Oliveira-Garcia and Hamilton 2024). Reason: Direct imaging of fungus-secreted Pwl2-mRFP in the extra-fungal BIC during infection. Supporting Evidence: PMID:38722963 Treatment with CLATHRIN HEAVY CHAIN inhibitor ES9β17 results in swollen, irregular localization of Pwl2:mRFP in BICs lacking obvious MEC puncta. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping of the UniProt subcellular location "Secreted", itself experimentally supported, to extracellular region. Reason: Correct mapping, consistent with the experimental annotations for the same term. Supporting Evidence: PMID:20435900 continued to accumulate in BICs after IH were growing elsewhere |
| GO:0030430 host cell cytoplasm | EXP PMID:20435900 Translocation of Magnaporthe oryzae effectors into rice cell... | ACCEPT | Summary: Fluorescent PWL2 was translocated from BICs into the rice cytoplasm and then moved into uninvaded neighbouring cells, in contrast to the apoplastic effector BAS4 which was not translocated (Khang et al. 2010). Reason: Foundational direct evidence for delivery into host cells. The host cytoplasm is where Pwl2 encounters its target HIPP43, so this is the core site of action. Supporting Evidence: PMID:20435900 were translocated into the rice cytoplasm PMID:20435900 proteins that reached the rice cytoplasm moved into uninvaded |
| GO:0030430 host cell cytoplasm | EXP PMID:26472068 Live-cell imaging of rice cytological changes reveals the im... | ACCEPT | Summary: Pwl2-mCherry was used as the symplastic (host-translocated) effector marker in live-cell imaging of rice infection (Mochizuki et al. 2015). Reason: The study treats Pwl2 as a symplastic effector and images it at BICs; consistent with the primary translocation evidence, so the curator's annotation is retained. Supporting Evidence: PMID:26472068 BICs were visualized using a fluorescently labeled symplastic effector, Pwl2:mCherry |
| GO:0030430 host cell cytoplasm | EXP PMID:30182232 Visualizing the Movement of Magnaporthe oryzae Effector Prot... | ACCEPT | Summary: Methods chapter describing assays that visualise PWL2 translocation into the rice cytoplasm and its cell-to-cell movement (abstract-only cache). Reason: The cached abstract explicitly describes assays for PWL2 translocation into the rice cytoplasm. Supporting Evidence: PMID:30182232 assays for visualizing M. oryzae effector translocation into the rice cytoplasm |
| GO:0030430 host cell cytoplasm | EXP PMID:31749824 Green Fluorescent Protein- and Discosoma sp. Red Fluorescent... | ACCEPT | Summary: Fungus-secreted PWL2-mCherry carrying a nuclear localization signal co-localised with the nuclear GFP-OsH2B marker in rice epidermal cells, confirming translocation into host cells (Chen et al. 2019). Reason: Host-cell entry is directly shown. The nuclear signal results from the engineered localization signal and does not indicate a native nuclear function, so host cell cytoplasm rather than host cell nucleus is the appropriate term. Supporting Evidence: PMID:31749824 oryzae effector PWL2 into host cells. |
| GO:0030430 host cell cytoplasm | EXP PMID:34236677 Characterizing the Secretion Systems of Magnaporthe oryzae. | ACCEPT | Summary: Protocol chapter characterising Pwl2 as a cytoplasmic (host-translocated) effector secreted by a nonconventional pathway, in contrast to the apoplastic Bas4 (abstract-only cache). Reason: The cached abstract classifies Pwl2 as a cytoplasmic effector, consistent with the primary translocation data. Supporting Evidence: PMID:34236677 two fluorescently labeled effectors, Bas4 (apoplastic) and Pwl2 (cytoplasmic) |
| GO:0030430 host cell cytoplasm | EXP PMID:38653755 Phytoalexin sakuranetin attenuates endocytosis and enhances ... | ACCEPT | Summary: PWL2-GFP expressed in transgenic rice is internalised into rice cells by clathrin-mediated endocytosis, and sakuranetin reduces this uptake (Jiang et al. 2024). Reason: A heterologous plant-expression system, but it supports entry of Pwl2 into the host cell interior via clathrin-mediated endocytosis, consistent with translocation observed during infection. Supporting Evidence: PMID:38653755 Some effectors, such as PWL2, are translocated into the rice cell cytoplasm via CME |
| GO:0030430 host cell cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic mapping of the UniProt subcellular location "Host cytoplasm", itself experimentally supported, to host cell cytoplasm. Reason: Correct mapping, consistent with the experimental annotations for the same term. Supporting Evidence: PMID:20435900 were translocated into the rice cytoplasm |
| GO:0052034 effector-mediated suppression of host pattern-triggered immunity | IDA PMID:40341381 The Magnaporthe oryzae effector Pwl2 alters HIPP43 localizat... | NEW | Summary: Stable barley lines expressing signal-peptide-less Pwl2-YFP show attenuated chitin- and flg22-induced reactive oxygen bursts and increased disease susceptibility; Pwl2 binds host HIPP43 and displaces it from plasmodesmata, and a non-binding Pwl2 variant neither displaces HIPP43 nor complements the reduced virulence of the triple deletion mutant (Were et al. 2025). Reason: GOA carries no process or function annotation for PWL2 at all, although its biological activity is now established. Participation test: the effector itself performs the step, binding the host target HIPP43 and relocalising it, and a binding-dead variant loses both the relocalisation and the virulence contribution, so this rests on more than necessity evidence. Comparator check: other secreted fungal effectors that suppress pattern-triggered immunity carry this symbiont-side branch (M. oryzae Slp1 and Cladosporium fulvum Ecp6 use the child term GO:0140423), confirming the branch is intended for effectors. The parent GO:0052034 rather than the signaling child GO:0140423 is proposed because the authors explicitly leave open whether HIPP43 acts within the immune signalling chain. Supporting Evidence: PMID:40341381 Furthermore, because Pwl2 attenuates the ROS burst in barley transgenic lines PMID:40341381 Pwl2 is a virulence factor that suppresses host immunity by perturbing the plasmodesmatal deployment of HIPP43 PMID:40341381 Finally, we show that a Pwl2SNDEYWY mutant unable to interact with HIPP43 fails to displace it from PDs and cannot complement the reduced virulence of Ξpwl2 mutants. PMID:38968126 OsHIPP43 interacted with Pwl2 with a calculated dissociation equilibrium constant (Kd) of 191 nM file:PYRO7/PWL2/PWL2-deep-research-falcon.md Its experimentally supported molecular activity is binding host proteins, particularly |
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Download this section (compressed HTML)Q: Which molecular-function term should capture Pwl2 binding to host HIPP43? Protein sequestering activity (GO:0140311) would fit displacement of HIPP43 from plasmodesmata, but the same study suggests Pwl2 may stabilise or increase HIPP43 accumulation, and HIPP43 overexpression alone suppresses the reactive oxygen burst, so it is unclear whether Pwl2 inactivates or co-opts its target. The same question applies to other MAX effectors that bind host small HMA proteins, such as AVR-Pik with OsHIPP19.
Q: Recognition of Pwl2 by barley MLA3 and by the unidentified weeping lovegrass resistance gene is a host-side event, and GO offers no symbiont-side term for being recognised. Should the avirulence and host-range role of PWL effectors be represented on the effector at all, or only through the plant receptor's annotations and GO-CAM models of the interaction?
Q: Would a cellular-component term for the biotrophic interfacial complex be useful? Cytoplasmic effectors such as Pwl2 and apoplastic effectors such as Bas4 are both annotated only to extracellular region, which loses the distinction between the two secretion systems and the two sites of action.
Experiment: Determine whether Pwl2 binding inhibits or stabilises HIPP43 in planta: measure HIPP43 protein half-life and plasmodesmatal pool with and without Pwl2, and test whether the Pwl2 variant that cannot bind HIPP43 behaves like the no-effector control, so that a sequestering versus stabilising molecular function can be assigned.
Experiment: Test whether HIPP43 acts upstream or downstream of reactive oxygen production by combining HIPP43 knockdown with chitin and flg22 elicitation and measuring receptor-complex assembly, so that the parent term (suppression of pattern-triggered immunity) can be refined to the signalling child term if warranted.
Experiment: Image native (untagged, antibody-detected) Pwl2 during rice infection to confirm that the plasmodesmatal relocalisation of HIPP43 observed in Nicotiana benthamiana and transgenic barley also occurs in the natural rice interaction.
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