MLO (Mildew resistance locus o) of barley encodes a 533-residue, plant-specific integral membrane protein that is the founder of the MLO family. It spans the plasma membrane seven times, with an extracellular N-terminus and a cytoplasmic C-terminal tail that carries a calmodulin-binding domain; calmodulin binds this domain in a Ca2+-dependent manner, and MLO-calmodulin association increases at fungal entry sites. Conserved extracellular cysteines, the second and third cytoplasmic loops and the C-terminal tail are required for activity, and MLO self-associates in planta. Wild-type MLO acts as a host susceptibility factor for the barley powdery mildew fungus Blumeria graminis f. sp. hordei: upon attack it is redistributed into a plasma membrane microdomain beneath fungal appressoria, where it dampens the cell-wall-restricted hydrogen peroxide burst and other defense responses that otherwise block fungal penetration of the leaf epidermis. Recessive loss-of-function mlo alleles therefore confer durable, broad-spectrum penetration resistance to powdery mildew (dependent on ROR1 and the syntaxin ROR2), accompanied by spontaneous mesophyll cell death and accelerated leaf senescence, and by enhanced susceptibility to some hemibiotrophic and necrotrophic fungi. MLO transcript levels rise in response to fungal attack, wounding, oxidative stress and senescence. The biochemical activity of barley MLO is not established; Arabidopsis MLO paralogs have been shown to act as Ca2+-permeable channels, making a channel function a leading hypothesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006952 defense response | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the Mlo domain (IPR004326). For barley MLO the sign of this annotation is inverted: wild-type MLO suppresses defense and promotes fungal entry, and resistance arises only when MLO is lost [PMID:9054509, PMID:12114562, PMID:11919636]. The Mlo domain mapping is also broad for the family as a whole, many of whose members act in pollen tube reception or root growth rather than defense [PMID:36588121]. Reason: An unqualified "defense response" implies that MLO executes defense, which is the opposite of what the barley genetics shows. Wild-type MLO dampens the penetration-site H2O2 burst and suppresses mesophyll oxidative burst and cell death, and the calmodulin-binding mutant halves MLO's ability to negatively regulate defense. "Negative regulation of defense response" (GO:0031348) captures the supported direction of effect and matches the term used for the Arabidopsis ortholog MLO2. A more specific "to fungus" child does not exist. The mechanism by which MLO suppresses defense is unknown, and the evidence is genetic (loss of function), so this is a regulatory, not an executive, process term; the more specific "defense response to fungus" is deliberately not proposed. Proposed replacements: negative regulation of defense response Supporting Evidence: PMID:12114562 Wild-type MLO dampens the cell wall-restricted hydrogen peroxide burst at points of attempted fungal penetration of the epidermal cell wall, and in subtending mesophyll cells, it suppresses a second oxidative burst and cell death. PMID:11919636 Loss of calmodulin binding halves the ability of MLO to negatively regulate defence against powdery mildew in vivo. PMID:9054509 The findings are compatible with a dual negative control function of the Mlo protein in leaf cell death and in the onset of pathogen defense; absence of Mlo primes the responsiveness for the onset of multiple defense functions. file:HORVU/MLO/MLO-deep-research-falcon.md the historical phrase “Mlo resistance” denotes resistance obtained by *losing* MLO activity, not an antimicrobial activity of the wild-type protein |
| GO:0016020 membrane | IEA GO_REF:0000120 | MODIFY | Summary: Correct but uninformative. Barley MLO is a seven-transmembrane integral membrane protein that was localized by leaf fractionation and immunoblotting to the plasma membrane [PMID:10574976], and live imaging shows it redistributing within the plasma membrane to fungal entry sites [PMID:15703292]. Reason: Direct experimental evidence places MLO in the plasma membrane, so the specific term should replace the generic one. Proposed replacements: plasma membrane Supporting Evidence: PMID:10574976 Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. PMID:15703292 fluorescently tagged MLO protein becomes redistributed in the plasma membrane (PM) and accumulates beneath fungal appressoria coincident with the initiation of pathogen entry into host cells |
| GO:0005516 calmodulin binding | IDA PMID:11919636 Calmodulin interacts with MLO protein to regulate defence ag... | NEW | Summary: MLO carries a C-terminal cytoplasmic domain that binds calmodulin in a Ca2+-dependent manner in vitro [PMID:11919636], and MLO-calmodulin FRET increases at fungal penetration sites in living barley cells [PMID:15703292]. Calmodulin binding is required for full MLO activity. Reason: This is a molecular function of MLO itself, demonstrated directly and functionally validated by mutagenesis of the binding domain (UniProt records L420 and W423 mutants). It is the only experimentally established molecular activity of barley MLO and is absent from GOA. Supporting Evidence: PMID:11919636 We have identified a domain in MLO that mediates a Ca2+-dependent interaction with calmodulin in vitro. PMID:15703292 We previously identified calmodulin, a cytoplasmic calcium sensor, as an interactor and positive regulator of MLO activity and demonstrate here by FRET microscopy an increase in MLO/calmodulin FRET around penetration sites coincident with successful host cell entry. |
Loading supporting content…
Download this section (compressed HTML)Q: Does barley MLO itself conduct Ca2+ (or other divalent cations) when expressed heterologously, as shown for Arabidopsis MLO1/5/9/15, and is channel activity required for powdery mildew susceptibility? If so, should calcium channel activity (GO:0005262) be added as the core molecular function?
Q: Is "negative regulation of defense response" the right process for a susceptibility factor whose mechanism is unknown, or would a qualifier such as acts_upstream_of_or_within (as TAIR uses for Arabidopsis MLO2) better reflect the loss-of-function basis of the evidence?
Experiment: Two-electrode voltage clamp or patch clamp of barley MLO in Xenopus oocytes or HEK cells, comparing wild-type MLO with resistance-conferring mlo point mutants and the calmodulin-binding mutant W423R, to test whether susceptibility function correlates with ion conductance.
Experiment: Genetically encoded Ca2+ reporter imaging at Blumeria penetration sites in Mlo versus mlo epidermal cells to determine whether MLO contributes to local Ca2+ influx during fungal entry.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)