MLO2 (MLO-like protein 2; At1g11310) is a 573-residue, plant-specific seven-transmembrane protein of the MLO family and the Arabidopsis co-ortholog (with MLO6 and MLO12, clade V) of barley MLO. It resides in the plasma membrane, with an extracellular N-terminus and a cytosolic C-terminal tail carrying a calmodulin-binding helix through which it associates with calmodulin (CAM2) in a Ca2+-dependent manner. Expressed in mammalian cells, MLO2 forms a divalent-cation-permeable channel that conducts Ca2+, Ba2+ and Mg2+ but not Na+ or K+, is blocked by La3+ and Gd3+, and is inhibited by co-expressed calmodulin. In leaf epidermal cells MLO2 is the major susceptibility factor for adapted powdery mildew fungi: upon attack it accumulates focally in the plasma membrane beneath fungal appressoria and around the penetration peg, and it enables fungal entry and suppression of host defenses, including indolic antimicrobial metabolites. mlo2 single mutants are partially resistant (resistance depends on PEN1, PEN2 and PEN3), while mlo2 mlo6 mlo12 triple mutants are fully penetration-resistant and mount faster, broader defense responses. Together with MLO6 and MLO12 and the exocyst subunit EXO70H4, MLO2 also contributes to polarized secretion and secondary cell wall formation in leaf trichomes. Whether its channel activity is what makes epidermal cells permissive to fungal entry is not yet established.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003674 molecular_function | ND GO_REF:0000015 | MODIFY | Summary: Root-term placeholder indicating no known molecular function. This is now outdated: MLO2 has been shown directly to conduct Ca2+ (and Ba2+, Mg2+) in heterologous patch-clamp experiments [PMID:35794475] and to bind calmodulin via its C-terminal calmodulin-binding domain [PMID:37767715]. Reason: Direct experimental evidence for MLO2 molecular activities exists, so the ND placeholder should be replaced by calcium channel activity and calmodulin binding (both also proposed as NEW annotations below). Proposed replacements: calcium channel activity calmodulin binding Supporting Evidence: PMID:35794475 To confirm Ca2+ imaging results, we used patch-clamp to directly measure transport activity of AtMLO2 and recorded large inward currents that depended on external Ca2+ concentrations PMID:37767715 found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions |
| GO:0005576 extracellular region | HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati... | REMOVE | Summary: High-throughput assignment from a size-exclusion/LC-MS profiling study of membrane-associated protein complexes in Arabidopsis leaves; MLO2 is not discussed individually. MLO2 is a polytopic integral membrane protein of the plasma membrane, with only short loops and the N-terminus facing the apoplast [file:ARATH/MLO2/MLO2-deep-research-falcon.md]. Reason: A seven-transmembrane integral plasma membrane protein is not a resident of the extracellular region; the assignment most likely reflects fraction classification in a large-scale proteomic dataset rather than localization of MLO2 outside the cell. Plasma membrane already captures the topologically correct location. Supporting Evidence: PMID:28887381 Here we combined cell fractionation of Arabidopsis leaves with nondenaturing detergent solubilization and LC/MS-based profiling of size exclusion chromatography fractions to measure the apparent masses of >1350 membrane-associated proteins. file:ARATH/MLO2/MLO2-deep-research-falcon.md MLO2 is a plant-specific, seven-transmembrane protein |
| GO:0005794 Golgi apparatus | HDA PMID:22430844 Isolation and proteomic characterization of the Arabidopsis ... | KEEP AS NON CORE | Summary: MLO2 was detected in a purified Golgi proteome. Live imaging of MLO2-GFP shows, in addition to the cell periphery, cytoplasmic puncta associated with a Golgi marker, consistent with MLO2 transiting the secretory pathway [file:ARATH/MLO2/MLO2-deep-research-falcon.md]. Reason: Plausible as a biosynthetic/trafficking location for a polytopic plasma membrane protein, and supported by imaging of Golgi-associated puncta, but the plasma membrane is where MLO2 acts. Supporting Evidence: PMID:22430844 A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome file:ARATH/MLO2/MLO2-deep-research-falcon.md Before challenge, MLO2–GFP has been observed at the cell periphery and in cytoplasmic puncta associated with a Golgi marker. |
| GO:0005886 plasma membrane | ISS PMID:16525893 Expression analysis of the AtMLO gene family encoding plant-... | ACCEPT | Summary: Inferred from barley MLO, which was biochemically shown to be a seven-transmembrane plasma membrane protein. Consistent with direct imaging of MLO2 fusions at the plasma membrane and in plasma membrane speckles in trichomes and at fungal attack sites [PMID:38124479]. Reason: Plasma membrane is the functional location of MLO2, where it acts as a Ca2+-permeable channel and accumulates beneath fungal appressoria. Supporting Evidence: PMID:16525893 The Arabidopsis (Arabidopsis thaliana) genome contains 15 genes encoding protein homologs of the barley mildew resistance locus o (MLO) protein biochemically shown to have a seven-transmembrane domain topology and localize to the plasma membrane. PMID:38124479 Here, MLO2-GFP co-localized with mCherry-EXO70H4 in PM speckles and the cell wall |
| GO:0005886 plasma membrane | TAS PMID:16525893 Expression analysis of the AtMLO gene family encoding plant-... | ACCEPT | Summary: Traceable author statement of plasma membrane localization for AtMLO proteins, based on barley MLO biochemistry; supported for MLO2 by later imaging [PMID:38124479]. Reason: Correct functional location of MLO2. Supporting Evidence: PMID:16525893 biochemically shown to have a seven-transmembrane domain topology and localize to the plasma membrane PMID:38124479 revealed that the MLO2-GFP fusion protein co-localized with mCherry-EXO70H4 in mature trichomes, mainly in the zone of the OR and above it at the PM delineating the thickened secondary cell wall |
| GO:0006952 defense response | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the Mlo domain. For MLO2 the sign is inverted: wild-type MLO2 enables powdery mildew entry and defense suppression, and resistance arises only when MLO2 (with MLO6 and MLO12) is lost [PMID:16732289, PMID:28674541, PMID:38124479]. Many MLO family members act in pollen tube reception or root growth, so the domain mapping is also broad at the family level [PMID:36588121]. Reason: An unqualified "defense response" implies MLO2 executes defense, the opposite of what loss-of-function genetics show. The triple mutant mounts an accelerated, broader defense response and the authors infer a role for MLO2/6/12 in enabling defense suppression. "Negative regulation of defense response" (GO:0031348) captures the supported direction of effect, is already annotated to MLO2 by TAIR from PMID:16732289, and matches the replacement used for barley MLO. The mechanism is unknown, so an executive defense term is deliberately not proposed. Proposed replacements: negative regulation of defense response Supporting Evidence: PMID:28674541 In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi. PMID:28674541 Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. |
| GO:0009506 plasmodesma | HDA PMID:21533090 Arabidopsis plasmodesmal proteome. | KEEP AS NON CORE | Summary: Detection in a cell-wall-derived plasmodesmal proteome. The authors note that this proteome is enriched for membrane proteins and contains contaminants; MLO2 was not validated individually. Reason: Plasmodesmata are lined by plasma membrane, so a plasma membrane protein being recovered here is plausible, but there is no targeted evidence that MLO2 is enriched at or functions in plasmodesmata. Supporting Evidence: PMID:21533090 Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: AtSubP sequence-based prediction. MLO2 is a seven-transmembrane plasma membrane protein; imaging places MLO2 fusions at the plasma membrane and in Golgi-associated puncta, and chloroplast-like signals seen near MLO2-GFP trichomes were attributed to underlying mesophyll cells [PMID:38124479]. Reason: Contradicted by experimental localization and topology; no chloroplast targeting evidence exists. Supporting Evidence: PMID:38124479 Note that the ellipsoid structures seen in the GFP channel in the case of the MLO2-GFP trichome likely are chloroplasts present in mesophyll cells beneath the trichome cell. |
| GO:0009620 response to fungus | IMP PMID:10677553 Isolation and characterization of powdery mildew-resistant A... | KEEP AS NON CORE | Summary: From the powdery mildew resistant (pmr) mutant screen; the pmr2 locus was later shown to correspond to MLO2 (cached abstract does not name MLO2, so the curator's full-text reading is deferred to). Loss of MLO2 alters the outcome of powdery mildew infection. Reason: A neutral, broad process term that correctly reflects that MLO2 function affects the plant-fungus interaction, but it describes a phenotype rather than what MLO2 does; the more informative term is negative regulation of defense response. Supporting Evidence: PMID:10677553 we identified 20 recessive Arabidopsis mutants that do not support normal growth of the powdery mildew pathogen, Erysiphe cichoracearum |
| GO:0016020 membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Correct but uninformative; MLO2 is a multi-pass membrane protein whose specific location (plasma membrane) is already annotated. Reason: True and non-misleading; the more specific plasma membrane annotation already exists, so no replacement is needed. Supporting Evidence: file:ARATH/MLO2/MLO2-deep-research-falcon.md MLO2 is a plant-specific, seven-transmembrane protein |
| GO:0031348 negative regulation of defense response | IMP PMID:16732289 Conserved requirement for a plant host cell protein in powde... | ACCEPT | Summary: Consonni et al. showed a conserved requirement for MLO proteins in powdery mildew pathogenesis in Arabidopsis; mlo2 resistance requires PEN1 (syntaxin), PEN2 (glycosyl hydrolase) and PEN3 (ABC transporter). Later work showed the mlo2 mlo6 mlo12 triple mutant mounts faster and broader defense responses and that the adapted fungus defeats indolic defense metabolite accumulation in an MLO-dependent manner [PMID:28674541]. Reason: Best available process term for MLO2: wild-type MLO2 suppresses defense outputs that otherwise block fungal entry. Consistent with the barley MLO review. Mechanism (possibly local Ca2+ influx) remains unresolved. Supporting Evidence: PMID:16732289 Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. PMID:16732289 Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. PMID:28674541 In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. |
| GO:0050832 defense response to fungus | IMP PMID:16732289 Conserved requirement for a plant host cell protein in powde... | MODIFY | Summary: Same paper and phenotype as the negative regulation of defense response row. Loss of MLO2 enhances penetration resistance to powdery mildew, i.e. wild-type MLO2 opposes rather than executes defense against the fungus [PMID:16732289, PMID:38124479]. Reason: The phenotype (resistance upon loss of function) establishes that MLO2 is a susceptibility factor, not a participant in defense. "Defense response to fungus" reads as MLO2 performing defense and is the necessity-vs-participation conflation flagged for MLO genes. The directionally correct term, negative regulation of defense response, is already supported by the same paper; this row should be replaced by it, as was done for barley MLO. Proposed replacements: negative regulation of defense response Supporting Evidence: PMID:38124479 Unlike mlo6 plants, mlo2-5 (∼64%) mutants supported significantly lower levels of host cell entry compared to Col-0, while mlo2-5 mlo6-2 mlo12-1 triple mutants were completely resistant (∼0%) PMID:28674541 In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi. |
| GO:0005262 calcium channel activity | IDA PMID:35794475 A receptor-channel trio conducts Ca(2+) signalling for polle... | NEW | Summary: AtMLO2 expressed in COS7/HEK293T cells mediated Ca2+ entry and large Ca2+-dependent inward currents in whole-cell patch clamp; it also conducted Ba2+ and Mg2+ but not K+ or Na+, and currents were blocked by La3+ and Gd3+. Co-expressed calmodulin strongly inhibited MLO-mediated Ca2+ entry, including for AtMLO2. Reason: Direct electrophysiological demonstration of channel activity of MLO2 itself (not a paralog), in a cell type lacking endogenous MLO. The family is now described as plant-specific Ca2+ channels [PMID:38124479]. Evidence is heterologous only; whether channel activity underlies powdery mildew susceptibility in planta is untested. Supporting Evidence: PMID:35794475 To confirm Ca2+ imaging results, we used patch-clamp to directly measure transport activity of AtMLO2 and recorded large inward currents that depended on external Ca2+ concentrations PMID:35794475 We also found that AtMLO2 was permeable to Ba2+ and Mg2+, but not to K+ or Na+ PMID:35794475 Furthermore, two typical Ca2+ channel blockers lanthanum (La3+) and gadolinium (Gd3+) inhibited the AtMLO2-mediated inward currents |
| GO:0005516 calmodulin binding | IDA PMID:37767715 Comprehensive comparative assessment of the Arabidopsis thal... | NEW | Summary: The MLO2 C-terminal calmodulin-binding domain interacts with CAM2 in most of seven independent in vitro and in vivo interaction assays; substitution of two key hydrophobic residues (LW/RR) reduced binding in most assays. In heterologous cells, calmodulin co-expression inhibits MLO2-mediated Ca2+ entry [PMID:35794475]. Reason: Direct, mutationally controlled evidence for a molecular interaction of MLO2 itself, with functional relevance (calmodulin feedback on channel activity). Matches the calmodulin binding annotation proposed for barley MLO. Supporting Evidence: PMID:37767715 We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. PMID:37767715 found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions PMID:35794475 A dramatic inhibition of Ca2+ entry was observed in all cases |
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Download this section (compressed HTML)Q: Is MLO2 channel activity required for powdery mildew susceptibility? Do resistance-conferring mlo2 point mutants lose Ca2+ conductance, and does local Ca2+ influx occur at penetration sites in an MLO2-dependent manner?
Q: Is "negative regulation of defense response" the best process term for a susceptibility factor whose mechanism is unknown, or is a term describing host-side facilitation of fungal entry (a host "susceptibility" process) needed in GO?
Q: Does MLO2 function in trichome secondary-wall secretion (with EXO70H4) independently of MLO6 and MLO12, or is this a strictly redundant clade V function?
Experiment: Patch clamp of wild-type MLO2 versus resistance-conferring mlo2 alleles and the LW/RR calmodulin-binding mutant in HEK293T cells, paired with complementation of mlo2 mlo6 mlo12 plants, to test whether conductance correlates with susceptibility.
Experiment: GCaMP or R-GECO Ca2+ imaging at Erysiphe/Golovinomyces penetration sites in wild type, mlo2 and mlo2 mlo6 mlo12 epidermal cells.
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