NFP (Nod Factor Perception) is a plasma-membrane LysM-domain receptor-like kinase (LysM-RLK) of the model legume Medicago truncatula. It is the essential, non-redundant receptor that perceives rhizobial lipo-chitooligosaccharide signals (Nod factors) secreted by Sinorhizobium meliloti, and triggers the earliest steps of the nitrogen-fixing root nodule symbiosis. The extracellular region carries three tandem, heavily N-glycosylated LysM domains that bind the chitin-based Nod factor ligand; a single transmembrane helix; and an intracellular protein-kinase-like domain. Critically, the NFP kinase domain is a catalytically dead PSEUDOKINASE: it deviates from conserved catalytic kinase residues and shows no autophosphorylation activity, so signal transduction requires association with an active co-receptor kinase, principally LYK3, with which NFP forms heteromeric receptor complexes at the cell periphery. NFP acts in both the root epidermis and cortex to drive Nod factor signalling (calcium spiking, early nodulin expression, root hair deformation), nodule organogenesis and rhizobial infection thread development/bacterial release. Beyond symbiosis, NFP also contributes to M. truncatula immunity against the oomycete Aphanomyces euteiches and the fungus Colletotrichum trifolii.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004713 protein tyrosine kinase activity | IEA GO_REF:0000043 | REMOVE | Summary: Retired SwissProt-keyword (SPKW) annotation derived from the "Tyrosine-protein kinase" keyword, which itself comes from the PROSITE PS00109 (PROTEIN_KINASE_TYR) signature match. This annotation is doubly incorrect. First, plant receptor-like kinases including NFP are serine/threonine kinases, not tyrosine kinases (UniProt itself names the protein "Serine/threonine receptor-like kinase NFP"). Second, and more fundamentally, the NFP intracellular kinase domain is a catalytically dead pseudokinase: it deviates from conserved catalytic residues and shows no detectable autophosphorylation activity. Reason: GOA's removal of this annotation was JUSTIFIED. The term is wrong on two counts. (1) NFP is a pseudokinase with no demonstrated catalytic activity: "NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases" and NFP belongs to "one subfamily ... characterized by deviations from conserved kinase sequences" (PMID:16844829). (2) Even if it were an active kinase, plant RLKs are Ser/Thr kinases; the tyrosine specificity is a PROSITE motif artefact, not a biological observation. The keyword2GO pipeline propagated a generic sequence-motif keyword without accounting for the experimentally established pseudokinase status. REMOVE; do not replace with any catalytic MF term. Supporting Evidence: PMID:16844829 Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. file:MEDTR/NFP/NFP-deep-research-falcon.md lacking key conserved motifs (including P-loop/DFG-related features and an activation-loop segment), and ... was observed in vitro, supporting interpretation of NFP as a likely ... or signaling subunit that requires an active kinase partner |
| GO:0006952 defense response | IEA GO_REF:0000043 | REMOVE | Summary: Retired SwissProt-keyword (SPKW) annotation derived from the "Plant defense" keyword. NFP does have a documented role in pathogen resistance (resistance to the oomycete Aphanomyces euteiches and the fungus Colletotrichum trifolii; PMID:23432463), so the keyword is not factually wrong. However, the broad "defense response" term adds nothing beyond the current GOA annotations, which already capture this role with the more specific and properly evidenced terms "positive regulation of defense response to oomycetes" (GO:1902290) and "regulation of defense response to fungus" (GO:1900150), both IMP from PMID:23432463. Reason: GOA's removal of this annotation was JUSTIFIED. The broad keyword-derived term is fully subsumed by the existing, more informative curated annotations GO:1902290 and GO:1900150 (both IMP, PMID:23432463), which are direct or indirect descendants of defense response and pinpoint the specific pathogen classes and regulatory nature of NFP's role. Retaining a generic IEA "defense response" alongside specific experimental terms is redundant over-annotation. NFP's primary, defining function is symbiotic Nod-factor perception; its defense role is real but secondary and already well represented. REMOVE. Supporting Evidence: PMID:23432463 nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A. euteiches, whereas NFP overexpression increased resistance. ... nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M. truncatula immunity. file:MEDTR/NFP/NFP-deep-research-falcon.md NFP/NFR-type receptors lie at a ... symbiosis–immunity interface ... Reviews summarize that NFs can induce transient defense outputs |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO IEA annotation from protein-kinase-domain signatures (IPR000719 Prot_kinase_dom, IPR001245 Ser-Thr/Tyr kinase catalytic domain, IPR008266 Tyr kinase active site). NFP does contain a protein-kinase-like domain in its intracellular region, but it is a catalytically dead pseudokinase: it lacks conserved catalytic residues and shows no autophosphorylation activity (PMID:16844829). The InterPro signatures detect the kinase FOLD but cannot distinguish active kinases from pseudokinases. Reason: Although less obviously wrong than the retired tyrosine-kinase keyword, "protein kinase activity" has the same underlying problem: it asserts a catalytic molecular function that NFP does not have. NFP is an experimentally established pseudokinase ("NFP did not show autophosphorylation activity ... deviations from conserved kinase sequences"; PMID:16844829), and signalling requires the active co-receptor kinase LYK3 (PMID:25351493). The kinase domain functions as a protein-interaction/scaffolding module (e.g. it binds the GTPase ROP10; PMID:25794934), not as an enzyme. An IEA term that contradicts direct experimental evidence should not be retained. REMOVE; the genuine molecular function (signalling receptor / Nod factor perception) is captured by the proposed NEW annotations below. Supporting Evidence: PMID:16844829 Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. PMID:25351493 our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules. file:MEDTR/NFP/NFP-deep-research-falcon.md lacking key conserved motifs (including P-loop/DFG-related features and an activation-loop segment), and ... was observed in vitro, supporting interpretation of NFP as a likely ... or signaling subunit that requires an active kinase partner |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO IEA annotation from the protein kinase domain signature (IPR000719). UniProt annotates a Gly-rich P-loop (residues 290-298) and a second ATP-binding residue (339) by PROSITE ProRule. Whether the pseudokinase domain of NFP actually binds ATP/nucleotide in vivo has not been tested; many pseudokinases retain nucleotide binding while others do not. Reason: The P-loop/VAIK motif elements predicted by sequence (UniProt BINDING 290-298 and 339) make residual ATP binding plausible even though NFP is catalytically inactive, and ATP binding by a pseudokinase does not imply catalysis. The annotation is not contradicted by experiment, but it is not a core function: NFP's defining role is ligand perception, not nucleotide chemistry, and no study has demonstrated functional ATP binding. Retain as a plausible domain-derived prediction, marked non-core. Supporting Evidence: PMID:16844829 one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. |
| GO:0005775 vacuolar lumen | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Subcellular-location IEA derived from the UniProt subcellular location "Vacuole lumen", which is itself based on the experimental observation that NFP relocalizes to the vacuolar lumen in nodule cells undergoing receptor breakdown (PMID:25351493). This IEA is the keyword/subcell-mapping counterpart of the curated IDA annotation below. Reason: The annotation accurately reflects the experimental finding that NFP is observed in the vacuolar lumen in cells undergoing receptor breakdown (PMID:25351493). It is a genuine but non-core, degradation-associated localization rather than the site of NFP signalling function. Keep, marked non-core (consistent with the curated IDA annotation from PMID:25351493). Supporting Evidence: UniProt:Q0GXS4 SUBCELLULAR LOCATION: Cell membrane; Single-pass membrane protein. Vacuole lumen. Note=Removed from the plasma membrane upon the release of rhizobia into the host cytoplasm. Vacuolar localization is observed in cells undergoing breakdown of the receptors. |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Multi-method IEA (ARBA + UniProtKB-SubCell mapping) placing NFP at the plasma membrane. NFP is a single-pass type I transmembrane protein and is experimentally localized at the cell periphery / plasma membrane (PMID:25351493). Reason: Correct and core: NFP is a plasma-membrane receptor. The localization is directly supported by experiment (curated IDA annotation from PMID:25351493) and by the protein's single-pass transmembrane topology. Accept as-is. Supporting Evidence: PMID:25351493 It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. |
| GO:0051707 response to other organism | IEA GO_REF:0000117 | MODIFY | Summary: ARBA machine-learning IEA. NFP responds to and perceives signals from other organisms - both the symbiotic rhizobium Sinorhizobium meliloti (Nod factors) and pathogenic oomycetes/fungi. The term is correct but very broad. Reason: "Response to other organism" is accurate but uninformatively general. The specific, experimentally documented responses are already, or should be, represented by more precise terms: response to molecule of bacterial origin (GO:0002237, existing IMP), nodulation (GO:0009877), and the defense-response regulation terms. The best single specific replacement that captures NFP's defining symbiotic role is "response to molecule of bacterial origin" (GO:0002237), already present from PMID:12753588. Proposed replacements: response to molecule of bacterial origin Supporting Evidence: PMID:12753588 The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. |
| GO:0005515 protein binding | IPI PMID:25794934 The small GTPase ROP10 of Medicago truncatula is required fo... | MODIFY | Summary: IPI annotation with WITH/FROM UniProtKB:B2MVQ1 (ROP10, a type II Rho-of-plants small GTPase). The kinase domain of NFP interacts with ROP10 in a GTP-dependent manner, and this interaction is required for root hair deformation during rhizobial infection (PMID:25794934). Reason: "Protein binding" (GO:0005515) is uninformative and discouraged. The interacting partner is a small GTPase and the interaction is well characterized (GTP-dependent, mediated by the NFP kinase domain), so a more specific molecular function term is warranted: "small GTPase binding" (GO:0031267). This also reframes the NFP kinase domain correctly - as a protein-interaction/scaffolding module rather than a catalytic kinase. Proposed replacements: small GTPase binding Supporting Evidence: PMID:25794934 ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. |
| GO:0009877 nodulation | IEP PMID:22874912 Epidermal and cortical roles of NFP and DMI3 in coordinating... | ACCEPT | Summary: IEP annotation: NFP expression is associated with nodulation. NFP is expressed in root epidermis and cortex and is required for nodule organogenesis; epidermal NFP alone is sufficient to induce cortical cell divisions leading to nodule primordia (PMID:22874912). Reason: Nodulation is the core biological process of NFP and is strongly supported. PMID:22874912 demonstrates that epidermal NFP induces cortical cell divisions leading to nodule primordia formation. Accept as a core function annotation. Supporting Evidence: PMID:22874912 Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation. |
| GO:0009877 nodulation | IMP PMID:22874912 Epidermal and cortical roles of NFP and DMI3 in coordinating... | ACCEPT | Summary: IMP annotation based on mutant/complementation analysis: tissue-specific complementation of nfp mutants shows epidermal NFP is sufficient to induce cortical cell divisions for nodule primordium formation (PMID:22874912). Reason: Strong genetic (loss-of-function plus tissue-targeted complementation) evidence that NFP is required for nodulation. Core function. Accept. Supporting Evidence: PMID:22874912 By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that ... Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation. |
| GO:1900150 regulation of defense response to fungus | IMP PMID:23432463 NFP, a LysM protein controlling Nod factor perception, also ... | ACCEPT | Summary: IMP annotation: nfp mutants show increased susceptibility to the fungus Colletotrichum trifolii, demonstrating that NFP positively contributes to antifungal immunity (PMID:23432463). Reason: Directly supported by mutant phenotype: "nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii" (PMID:23432463). This is a specific, well-evidenced secondary (non-core) function distinct from NFP's primary symbiotic role. Accept. (The data arguably support the more specific "positive regulation of defense response to fungus", but the parent regulatory term as annotated is correct and adequately specific.) Supporting Evidence: PMID:23432463 nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M. truncatula immunity. |
| GO:1902290 positive regulation of defense response to oomycetes | IMP PMID:23432463 NFP, a LysM protein controlling Nod factor perception, also ... | ACCEPT | Summary: IMP annotation: nfp mutants are more susceptible to the oomycete Aphanomyces euteiches, and NFP overexpression increases resistance, establishing NFP as a positive regulator of anti-oomycete defense (PMID:23432463). Reason: Well supported by reciprocal genetic evidence: loss of function increases susceptibility while overexpression increases resistance to A. euteiches (PMID:23432463). The "positive regulation" directionality is justified by the overexpression result. Specific, properly evidenced secondary function. Accept. Supporting Evidence: PMID:23432463 nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A. euteiches, whereas NFP overexpression increased resistance. |
| GO:0002237 response to molecule of bacterial origin | IMP PMID:12753588 The NFP locus of Medicago truncatula controls an early step ... | ACCEPT | Summary: IMP annotation: nfp mutants fail to respond to rhizobial Nod factors (lipo-chitooligosaccharides of bacterial origin) by any assayed response - no calcium flux, no calcium spiking, no early nodulin expression, no root hair deformation (PMID:12753588). Reason: Core function. The nfp mutant is completely unresponsive to the rhizobial Nod factor, establishing NFP as essential for perception of and response to this bacterial-origin molecule (PMID:12753588). Accept; this term also serves as the specific replacement for the broad ARBA term "response to other organism". Supporting Evidence: PMID:12753588 The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. |
| GO:0009101 glycoprotein biosynthetic process | IDA PMID:16723404 LysM domains of Medicago truncatula NFP protein involved in ... | MARK AS OVER ANNOTATED | Summary: IDA annotation based on PMID:16723404, which showed that NFP expressed in M. truncatula roots is highly N-glycosylated, probably with both high-mannose and complex glycans. This annotation describes NFP as a glycoprotein substrate, not as an enzyme of the glycosylation machinery. Reason: NFP is the SUBSTRATE of N-glycosylation, not a component of the glycoprotein biosynthetic pathway. Being a glycoprotein does not mean the gene product is "involved in" the biosynthetic process in the causal sense that the GO BP term implies. The underlying observation (NFP is N-glycosylated) is better captured as a protein modification/feature (UniProt CARBOHYD features) than as a BP annotation. This is an over-annotation: NFP plays no role in synthesizing glycans. Mark as over-annotated. Supporting Evidence: PMID:16723404 Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. |
| GO:0009877 nodulation | IMP PMID:12753588 The NFP locus of Medicago truncatula controls an early step ... | ACCEPT | Summary: IMP annotation: the nfp mutant has a Nod-negative phenotype, failing all Nod factor responses and unable to nodulate (PMID:12753588). NFP controls the earliest, nodulation-specific step of Nod factor signal transduction. Reason: Core function with strong loss-of-function evidence. The nfp mutant is Nod-negative and NFP "controls an early step of Nod factor signal transduction ... specific to nodulation" (PMID:12753588). Accept. Supporting Evidence: PMID:12753588 These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation. |
| GO:0009877 nodulation | IEP PMID:16844829 The Medicago truncatula lysin [corrected] motif-receptor-lik... | ACCEPT | Summary: IEP annotation: NFP is expressed in association with infection thread development and nodule formation, and is involved in the infection process (PMID:16844829). Reason: Expression-based support for the core nodulation function; NFP is expressed in roots and nodules in association with infection thread development (PMID:16844829). Consistent with the IMP annotations. Accept. Supporting Evidence: PMID:16844829 NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. |
| GO:0009877 nodulation | IMP PMID:16844829 The Medicago truncatula lysin [corrected] motif-receptor-lik... | ACCEPT | Summary: IMP annotation: nfp-1 mutant analysis shows impaired nodulation - no root hair curling, no infection thread formation, no Nod factor-induced root hair deformation (PMID:16844829). Reason: Core function, strong mutant evidence. The nfp-1 mutant shows an impaired nodulation response to S. meliloti (PMID:16844829). Accept. Supporting Evidence: PMID:16844829 NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. |
| GO:0005515 protein binding | IPI PMID:25351493 Nod factor receptors form heteromeric complexes and are esse... | MODIFY | Summary: IPI annotation with WITH/FROM UniProtKB:Q6UD73 (LYK3, an active LysM receptor-like kinase). FRET-FLIM analysis showed that NFP and LYK3 form heteromeric receptor complexes at the cell periphery in M. truncatula nodules (PMID:25351493). Reason: "Protein binding" (GO:0005515) is uninformative and discouraged. The characterized interaction is a specific heteromeric receptor complex between two LysM-RLKs, so a more informative molecular function term is appropriate: "protein heterodimerization activity" (GO:0046982). This interaction is functionally central - because NFP is a pseudokinase, the heteromer with the active kinase LYK3 is the mechanism by which Nod factor perception is transduced. Proposed replacements: protein heterodimerization activity Supporting Evidence: PMID:25351493 our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules. |
| GO:0005775 vacuolar lumen | IDA PMID:25351493 Nod factor receptors form heteromeric complexes and are esse... | KEEP AS NON CORE | Summary: IDA annotation: NFP is observed in the vacuolar lumen of nodule cells undergoing breakdown of the receptors (PMID:25351493). Reason: Directly observed localization, but it represents a degradation/turnover route ("Vacuolar localization is observed in cells undergoing breakdown of the receptors") rather than the site of NFP's signalling function. Keep, but mark non-core; the functionally relevant localization is the plasma membrane. Supporting Evidence: PMID:25351493 the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. |
| GO:0005886 plasma membrane | IDA PMID:25351493 Nod factor receptors form heteromeric complexes and are esse... | ACCEPT | Summary: IDA annotation: NFP localizes at the cell periphery / plasma membrane in a narrow zone of about two cell layers at the nodule apex, where it can perceive bacterial Nod factors (PMID:25351493). Reason: Core localization, directly observed. NFP is a plasma-membrane receptor; the plasma membrane is where it perceives Nod factors and forms heteromeric complexes with LYK3 (PMID:25351493). Accept. Supporting Evidence: PMID:25351493 It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. |
| GO:0009877 nodulation | IEP PMID:25351493 Nod factor receptors form heteromeric complexes and are esse... | ACCEPT | Summary: IEP annotation: NFP expression/protein accumulation is associated with nodulation, specifically with the meristem and infection zone of developing nodules where it perceives Nod factors (PMID:25351493). Reason: Expression-pattern support for the core nodulation function, consistent with the multiple IMP annotations. Accept. Supporting Evidence: PMID:25351493 In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. |
| GO:0038023 signaling receptor activity | IMP PMID:12753588 The NFP locus of Medicago truncatula controls an early step ... | NEW | Summary: NFP is a Nod factor receptor: its LysM ectodomain binds the rhizobial lipo-chitooligosaccharide signal and the receptor triggers intracellular signalling. The nfp mutant fails every Nod factor response, placing NFP at the most upstream, receptor-level step of the pathway. Reason: The current GOA set lacks any molecular function term for NFP's true activity. With the catalytic kinase terms removed (NFP is a pseudokinase), a signalling receptor MF term is needed to represent the gene's defining function. "Signaling receptor activity" (GO:0038023) is well supported: the nfp mutant abolishes all Nod factor responses (PMID:12753588) and NFP is described as a Nod factor receptor that perceives the bacterial signal (PMID:25351493, PMID:16723404). Supporting Evidence: PMID:12753588 These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation. PMID:25351493 In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. |
| GO:0008061 chitin binding | ISS PMID:16723404 LysM domains of Medicago truncatula NFP protein involved in ... | NEW | Summary: The NFP extracellular region contains three tandem LysM domains. LysM domains are carbohydrate-binding modules that bind N-acetylglucosamine polymers (chitin/chitooligosaccharides); the rhizobial Nod factor is a lipo-chitooligosaccharide. Homology modelling and docking predict that the Nod factor / chitooligosaccharide binds the three LysM domains, and LysM residues (e.g. Leu154) are functionally required for Nod factor recognition. Reason: NFP's molecular recognition function - binding the chitin-based Nod factor ligand via its LysM domains - is central and currently unrepresented in GOA. "Chitin binding" (GO:0008061) is the closest available GO term for the chitooligosaccharide/LCO-binding activity of the LysM ectodomain. Supported by modelling/docking (PMID:16723404) and by mutagenesis showing LysM residues are required for Nod factor recognition (PMID:22087221). Evidence is structural-modelling/inference plus mutant data, hence ISS. Supporting Evidence: PMID:16723404 A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. PMID:22087221 we have demonstrated the importance of the NFP LysM2 domain for rhizobial infection and we have pinpointed the importance of a single leucine residue of LysM2 in that step of the symbiosis. |
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Download this section (compressed HTML)Q: Does the NFP pseudokinase domain retain ATP/nucleotide binding in vitro, or has it lost nucleotide binding as well as catalysis?
Q: What is the structural basis of Nod factor recognition by the NFP LysM ectodomain, and which LysM domain(s) directly contact the lipo-chitooligosaccharide acyl and sulphate substitutions?
Q: How is signal transferred from the NFP pseudokinase domain to LYK3 within the heteromeric complex - by allosteric activation, transphosphorylation of NFP by LYK3, or scaffolding of downstream effectors?
Q: Does NFP perception of pathogen-derived chitooligosaccharides use the same LysM binding surface as Nod factor perception, and how is the symbiotic versus immune signalling outcome discriminated?
Experiment: Reconstitute the NFP/LYK3 complex in vitro and test whether the active LYK3 kinase transphosphorylates the NFP pseudokinase domain, and whether NFP allosterically modulates LYK3 activity.
Hypothesis: NFP is a catalytically dead pseudokinase that is transphosphorylated by and allosterically regulates its active co-receptor LYK3.
Experiment: Solve a co-crystal or cryo-EM structure of the NFP LysM ectodomain bound to a defined Sinorhizobium meliloti Nod factor to confirm the predicted three-LysM-domain binding mode.
Hypothesis: The rhizobial lipo-chitooligosaccharide Nod factor binds across the three tandem LysM domains of the NFP ectodomain.
Experiment: Quantitative binding assays (ITC/SPR) with purified NFP ectodomain against Nod factors, chitooligosaccharides of varying length, and pathogen-derived chitin fragments to define ligand specificity and affinity.
Hypothesis: The NFP LysM ectodomain binds chitin-based ligands and discriminates symbiotic Nod factors from pathogen-derived chitooligosaccharides by affinity or substitution recognition.
Experiment: Structure-guided mutagenesis of NFP pseudokinase-domain residues to test whether nucleotide binding or specific surface residues are required for ROP10 interaction and for signalling output.
Hypothesis: The NFP pseudokinase domain functions as a protein-interaction module whose surface, rather than catalytic activity, is required for ROP10 binding and signalling.
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