Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0004672 protein kinase activity	IEA GO_REF:0000002	MODIFY	Summary: BIK1 is a catalytically active protein kinase; this InterPro-based electronic annotation is correct but less specific than the experimentally supported Ser/Thr kinase terms. Reason: Correct but general. BIK1 is a proven Ser/Thr (dual-specificity) protein kinase (EC 2.7.11.1); the more specific term protein serine/threonine kinase activity is preferred and is independently supported by experimental annotations. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:24104392 we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time.
GO:0004674 protein serine/threonine kinase activity	IEA GO_REF:0000003	ACCEPT	Summary: Electronic EC-based annotation of Ser/Thr protein kinase activity; correct and a core molecular function of BIK1. Reason: BIK1 is an experimentally validated Ser/Thr protein kinase (EC 2.7.11.1) that phosphorylates RBOHD, OSCA1.3, FLS2, BAK1 and WRKY transcription factors. The electronic EC mapping correctly captures the core catalytic function. Supporting Evidence: PMID:32846426 This phosphorylation is dependent on BIK1 kinase activity, since a kinase-dead variant, GST–BIK1(KD), did not phosphorylate OSCA1.3-loop1
GO:0005524 ATP binding	IEA GO_REF:0000002	ACCEPT	Summary: ATP binding is required for BIK1 kinase catalysis; supported by a defined ATP-binding site and an ATP-binding Lys-105 whose mutation abolishes activity. Reason: BIK1 has a canonical protein kinase ATP-binding pocket (Gly-rich loop 73-81, catalytic Lys-105). Mutation of Lys-105 (with Lys-106) gives a kinase-dead enzyme, confirming functional ATP binding. Standard supporting MF for an active kinase. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md ATP-binding Lys-105; active-site (proton acceptor) Asp-202.
GO:0005634 nucleus	IEA GO_REF:0000044	ACCEPT	Summary: Nuclear localization is supported by a focused study showing activation-dependent relocation of BIK1 to the nucleus; this electronic subcellular-location annotation is corroborated experimentally. Reason: A pool of phosphorylated/activated BIK1 moves to the nucleus and phosphorylates WRKY transcription factors. Although BIK1 is predominantly plasma membrane-anchored, nuclear localization is a genuine, mechanistically defined secondary location. Supporting Evidence: PMID:29649442 BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation.
GO:0005886 plasma membrane	IEA GO_REF:0000044	ACCEPT	Summary: Plasma membrane is the principal location of BIK1; this electronic annotation is strongly supported by lipid-anchoring and multiple focused studies. Reason: BIK1 is myristoylated (Gly-2) and palmitoylated (Cys-4) and resides at the plasma membrane where it associates with PRR complexes. Core location for its immune signaling function. Supporting Evidence: PMID:16339855 BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response.
GO:0010008 endosome membrane	IEA GO_REF:0000044	KEEP AS NON CORE	Summary: BIK1 is internalized into endocytic compartments after ligand-induced monoubiquitination; endosome membrane localization is experimentally supported. Reason: Endosomal localization reflects ligand-induced internalization of BIK1 with FLS2, a regulated trafficking step downstream of activation rather than the primary site of catalytic immune signaling. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md supported by ligand-induced internalization study
GO:0106310 protein serine kinase activity	IEA GO_REF:0000116	ACCEPT	Summary: Rhea/EC-based electronic annotation of protein serine kinase activity; consistent with experimentally proven BIK1 catalytic activity. Reason: BIK1 phosphorylates serine residues on substrates such as RBOHD (S39/S339/ S343) and OSCA1.3 (S54). The electronic annotation correctly captures part of the core kinase activity; it is independently supported by EXP annotations. Supporting Evidence: PMID:32846426 BIK1 predominantly phosphorylates S54
GO:0005515 protein binding	IPI PMID:20018686 A receptor-like cytoplasmic kinase, BIK1, associates with a ...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from the BIK1-FLS2/BAK1 interaction study; uninformative as a molecular function although the interaction itself is real and central. Reason: Per curation guidelines, bare GO:0005515 protein binding is uninformative. The underlying interaction (BIK1 associating with and trans-phosphorylating the FLS2/BAK1 receptor complex) is genuine and is captured by the kinase activity MF and the PRR signaling BP terms; the bare binding term should not be treated as a core function. Supporting Evidence: PMID:20018686 BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro.
GO:0004674 protein serine/threonine kinase activity	TAS PMID:30584105 Conserved fungal effector suppresses PAMP-triggered immunity...	ACCEPT	Summary: Traceable assertion of Ser/Thr protein kinase activity; correct and a core molecular function of BIK1. Reason: BIK1 is an experimentally validated Ser/Thr protein kinase. This is one of several annotations capturing the core catalytic function and is well supported. Supporting Evidence: PMID:24104392 Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity. file:ARATH/BIK1/BIK1-deep-research-falcon.md Experimental data support BIK1 as an active serine/threonine protein kinase
GO:0005886 plasma membrane	EXP PMID:26021844 Regulatory role of BOTRYTIS INDUCED KINASE1 in ETHYLENE INSE...	ACCEPT	Summary: Experimental support for plasma membrane localization via myristoylation at Gly-2; core location. Reason: This study mapped N-myristoylation at Gly-2 and showed that Gly-2 mutation drastically reduces plasma membrane localization, directly demonstrating lipid-anchored PM residence. Core to BIK1 function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md Drastic reduction of plasma membrane localization and strong increase of cytoplasmic localization.
GO:0005886 plasma membrane	EXP PMID:32404997 Ligand-induced monoubiquitination of BIK1 regulates plant im...	ACCEPT	Summary: Experimental support for plasma membrane localization where BIK1 resides with FLS2 before ligand-induced internalization; core location. Reason: BIK1 is monoubiquitinated and internalized from the plasma membrane upon flg22 perception, establishing the PM as the resting site of BIK1 within the receptor complex. Core location. Supporting Evidence: PMID:32404997 Ligand-induced monoubiquitination of BIK1 regulates plant immunity.
GO:0010008 endosome membrane	EXP PMID:32404997 Ligand-induced monoubiquitination of BIK1 regulates plant im...	KEEP AS NON CORE	Summary: Experimentally supported endosome membrane localization following ligand-induced internalization; a regulated downstream trafficking step. Reason: BIK1 internalizes into endocytic compartments after monoubiquitination upon flg22 perception. Real but a downstream regulatory localization, not the primary site of signaling activity. Supporting Evidence: PMID:32404997 Ligand-induced monoubiquitination of BIK1 regulates plant immunity.
GO:0106310 protein serine kinase activity	EXP PMID:24104392 Identification and functional analysis of phosphorylation re...	ACCEPT	Summary: Experimentally demonstrated protein serine kinase activity (and dual-specificity autophosphorylation); core molecular function. Reason: This study profiled BIK1 autophosphorylation, demonstrating serine phosphorylation activity and establishing BIK1 as a dual-specificity kinase. Core catalytic function. Supporting Evidence: PMID:24104392 we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time.
GO:0106310 protein serine kinase activity	EXP PMID:32846426 The calcium-permeable channel OSCA1.3 regulates plant stomat...	ACCEPT	Summary: Experimentally demonstrated serine kinase activity toward OSCA1.3 (S54); core molecular function. Reason: BIK1 directly phosphorylates the OSCA1.3 channel at Ser-54 in a kinase-activity-dependent manner, directly demonstrating protein serine kinase activity on a physiological substrate. Core catalytic function. Supporting Evidence: PMID:32846426 BIK1 predominantly phosphorylates S54
GO:0005515 protein binding	IPI PMID:31803215 BIK1 and ERECTA Play Opposing Roles in Both Leaf and Inflore...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from the BIK1-ERECTA developmental study; uninformative MF although the interaction (and phosphorylation of ER) is real. Reason: Bare GO:0005515 is uninformative. The specific relationship (BIK1 interacts with and phosphorylates ERECTA-family RLKs in leaf/inflorescence development) is a genuine kinase-substrate interaction captured by the kinase MF; the bare binding term is not a core function. Supporting Evidence: PMID:31803215 BIK1 interacts with ER-family proteins and directly phosphorylates ER.
GO:0005634 nucleus	HDA PMID:15610358 High-throughput protein localization in Arabidopsis using Ag...	ACCEPT	Summary: Nucleus from a generic high-throughput GFP-ORF screen; the location is independently corroborated by a focused activation-dependent study so it is accepted. Reason: Although this HDA call comes from a generic GFP-ORF overexpression localization screen rather than a focused BIK1 study, nuclear localization is independently and directly demonstrated (PMID:29649442) as a regulated, activation-dependent location where BIK1 phosphorylates WRKY TFs. Supporting Evidence: PMID:29649442 BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors
GO:0005730 nucleolus	HDA PMID:15610358 High-throughput protein localization in Arabidopsis using Ag...	MARK AS OVER ANNOTATED	Summary: Nucleolar localization derives only from a generic GFP-ORF overexpression screen and is not substantiated by any focused BIK1 study; likely an over-annotation. Reason: The nucleolus call comes from a high-throughput GFP-ORF transient overexpression screen that classified proteins into broad categories including nucleolar. It is biologically implausible for a myristoylated/palmitoylated, membrane-anchored immune kinase and is not corroborated by any dedicated BIK1 study (focused work supports plasma membrane, nucleus and endosomes only). Supporting Evidence: PMID:15610358 These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments.
GO:0005737 cytoplasm	HDA PMID:15610358 High-throughput protein localization in Arabidopsis using Ag...	KEEP AS NON CORE	Summary: Cytoplasm from a generic GFP-ORF overexpression screen; weakly supported and likely an artifact of overexpression, kept as non-core. Reason: The cytoplasmic HDA call is from a generic overexpression localization screen. A cytoplasmic pool can appear when membrane targeting (myristoylation/ palmitoylation) is overwhelmed or disrupted (e.g. Gly-2 mutation increases cytoplasmic signal), so it is biologically plausible but not a core functional location; retained as non-core. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md strong increase of cytoplasmic localization
GO:0004674 protein serine/threonine kinase activity	IDA PMID:32846426 The calcium-permeable channel OSCA1.3 regulates plant stomat...	ACCEPT	Summary: Direct experimental demonstration of Ser/Thr protein kinase activity (phosphorylation of OSCA1.3); core molecular function. Reason: In vitro radioactive kinase assays show BIK1 directly phosphorylates OSCA1.3 in a kinase-activity-dependent manner, directly demonstrating the core Ser/Thr kinase function. Supporting Evidence: PMID:32846426 This phosphorylation is dependent on BIK1 kinase activity, since a kinase-dead variant, GST–BIK1(KD), did not phosphorylate OSCA1.3-loop1
GO:0006468 protein phosphorylation	IDA PMID:32846426 The calcium-permeable channel OSCA1.3 regulates plant stomat...	ACCEPT	Summary: Protein phosphorylation as the biological process by which BIK1 transmits immune signals (e.g. phosphorylating OSCA1.3); core activity. Reason: BIK1 transduces immune signals by phosphorylating downstream substrates. The BP term protein phosphorylation correctly captures this; directly supported by OSCA1.3 phosphorylation. Supporting Evidence: PMID:32846426 BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22
GO:0010119 regulation of stomatal movement	IGI PMID:32846426 The calcium-permeable channel OSCA1.3 regulates plant stomat...	ACCEPT	Summary: BIK1 controls stomatal closure during immunity via phosphorylation of the OSCA1.3 Ca2+ channel; well-supported regulatory role. Reason: BIK1-mediated phosphorylation of OSCA1.3 is required for flg22-induced stomatal closure (stomatal immunity), and BIK1/RBOHD also controls stomatal movement. A genuine, mechanistically defined role in regulating stomatal movement in the immune context. Supporting Evidence: PMID:32846426 OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling file:ARATH/BIK1/BIK1-deep-research-falcon.md BIK1 (with PBL1) is required for MAMP/DAMP-induced calcium elevations, positioning BIK1 in early signaling outputs beyond ROS.
GO:0005515 protein binding	IPI DOI:10.1038/s41586-020-2210-3	MARK AS OVER ANNOTATED	Summary: Bare protein binding from the monoubiquitination study (BIK1 binds FLS2 and the ATL44/ATL45 ubiquitin ligases); uninformative MF. Reason: Bare GO:0005515 is uninformative. The underlying interactions (BIK1 with FLS2 and with the RING E3 ligases ATL44/RHA3A and ATL45/RHA3B) are real and are captured by the PRR signaling and ubiquitination-related biology described in the notes; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md monoubiquitination of BIK1 by ATL44/RHA3A and ATL45/RHA3B
GO:0012505 endomembrane system	IDA DOI:10.1038/s41586-020-2210-3	KEEP AS NON CORE	Summary: Endomembrane system localization reflecting ligand-induced internalization of BIK1; correct but more specific endosome terms are also annotated. Reason: Consistent with BIK1 internalization into endocytic compartments after monoubiquitination. The more specific endosome/endosome membrane terms are also present; this general term is retained as a non-core, downstream trafficking location. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md supported by ligand-induced internalization study
GO:0002221 pattern recognition receptor signaling pathway	IMP DOI:10.1038/s41586-020-2210-3	ACCEPT	Summary: BIK1 is a central component of PRR (FLS2/EFR-BAK1) signaling; core biological process. Reason: BIK1 is the canonical RLCK relaying signaling from cell-surface PRR complexes downstream of PAMP perception. PRR signaling pathway is a core BP for BIK1, supported by loss-of-function phenotypes in immune signaling. Supporting Evidence: PMID:20018686 BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling. file:ARATH/BIK1/BIK1-deep-research-falcon.md BIK1 is repeatedly described as a PRR-associated kinase acting downstream of multiple PRRs (FLS2, EFR; also CERK1, PEPR1), linking PAMP perception to downstream defense signaling.
GO:0002237 response to molecule of bacterial origin	IDA DOI:10.1038/s41586-020-2210-3	ACCEPT	Summary: BIK1 mediates responses to bacterial PAMPs such as flagellin (flg22); core immune process. Reason: BIK1 is phosphorylated and monoubiquitinated specifically in response to the bacterial PAMP flg22 and is required for the downstream immune response. The term (which explicitly covers flagellin-derived peptides) is a core BP for BIK1. Supporting Evidence: PMID:20018686 BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1.
GO:0005768 endosome	IDA DOI:10.1038/s41586-020-2210-3	KEEP AS NON CORE	Summary: Endosome localization from ligand-induced internalization of BIK1; experimentally supported downstream trafficking location. Reason: BIK1 internalizes into endosomes after monoubiquitination upon flg22 perception. A genuine but downstream regulatory localization, not the primary site of signaling activity. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md internalization "into endocytic compartments"
GO:0005886 plasma membrane	IDA DOI:10.1038/s41586-020-2210-3	ACCEPT	Summary: Plasma membrane localization of BIK1 within the FLS2 complex prior to internalization; core location. Reason: BIK1 resides at the plasma membrane in the resting receptor complex and is internalized upon ligand perception, confirming the PM as its primary functional location. Core. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md Plasma membrane is the principal, well-supported location
GO:0042742 defense response to bacterium	IMP DOI:10.1038/s41586-020-2210-3	ACCEPT	Summary: BIK1 is required for defense against bacterial pathogens (e.g. Pseudomonas syringae); core immune function. Reason: BIK1 ubiquitination mutants show enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000, and BIK1 drives the ROS/Ca2+ outputs that restrict bacterial entry. Defense response to bacterium is a core BP. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md negative regulator of basal resistance to Pst file:ARATH/BIK1/BIK1-deep-research-falcon.md BIK1-dependent signaling through RBOHD is linked to stomatal movement that restricts bacterial entry, with impairment when BIK1 function (or its RBOHD phosphorylation) is compromised.
GO:0050832 defense response to fungus	IMP DOI:10.1038/s41586-020-2210-3	ACCEPT	Summary: BIK1 contributes to defense against fungal pathogens (e.g. Botrytis cinerea); supported immune function. Reason: BIK1 was originally identified as required for resistance to necrotrophic fungi, and ubiquitination mutants show enhanced susceptibility to Botrytis cinerea. Defense response to fungus is a genuine, core-adjacent immune BP. Supporting Evidence: PMID:16339855 Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens
GO:0005634 nucleus	IDA PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	ACCEPT	Summary: Direct experimental evidence that activated BIK1 localizes to the nucleus to phosphorylate WRKY TFs; mechanistically defined secondary location. Reason: This focused study directly demonstrated nuclear localization of BIK1 and its interaction with nuclear WRKY transcription factors regulating JA/SA. A genuine activation-dependent location supporting the PRR-BIK1-WRKY axis. Supporting Evidence: PMID:29649442 BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation.
GO:0005886 plasma membrane	IDA PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	ACCEPT	Summary: Direct experimental confirmation of plasma membrane localization (the documented resting location of BIK1); core location. Reason: This study confirms BIK1's documented plasma membrane localization alongside its activation-dependent nuclear pool. PM is the core functional location. Supporting Evidence: PMID:29649442 in addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus
GO:0080141 regulation of jasmonic acid biosynthetic process	IEP PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	KEEP AS NON CORE	Summary: BIK1 regulates JA levels via phosphorylation of WRKY TFs; a downstream regulatory output rather than the core biochemical function. Reason: BIK1 modulates JA homeostasis through the PRR-BIK1-WRKY axis. This is a genuine but downstream/regulatory consequence of BIK1 immune signaling, not its core kinase function; retained as non-core. Supporting Evidence: PMID:29649442 EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1.
GO:0080141 regulation of jasmonic acid biosynthetic process	IMP PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	KEEP AS NON CORE	Summary: BIK1 phosphosite mutants alter JA levels, supporting a regulatory role in JA biosynthesis; downstream output, non-core. Reason: Phosphomimetic mutations at EFR-targeted BIK1 sites elevate JA, genetically linking BIK1 to JA regulation. Genuine but a downstream regulatory output of BIK1 signaling, not its core function. Supporting Evidence: PMID:29649442 Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections.
GO:0080142 regulation of salicylic acid biosynthetic process	IEP PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	KEEP AS NON CORE	Summary: BIK1 regulates SA homeostasis via the PRR-BIK1-WRKY axis; downstream regulatory output, non-core. Reason: BIK1 modulates SA levels through phosphorylation of WRKY transcription factors. A genuine downstream regulatory consequence of BIK1 signaling, retained as non-core. (BIK1 was also originally shown to influence SA accumulation.) Supporting Evidence: PMID:29649442 WRKY transcription factors involved in the JA and salicylic acid (SA) regulation
GO:0080142 regulation of salicylic acid biosynthetic process	IMP PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	KEEP AS NON CORE	Summary: BIK1 phosphosite mutants alter SA-associated defense outputs; downstream regulatory role, non-core. Reason: Mutations at EFR-targeted BIK1 phosphosites change hormone levels and bacterial resistance, genetically linking BIK1 to SA regulation. Downstream output of BIK1 signaling, retained as non-core. Supporting Evidence: PMID:29649442 Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections.
GO:0002237 response to molecule of bacterial origin	IMP PMID:25522736 Microbe-associated molecular pattern-induced calcium signali...	ACCEPT	Summary: BIK1 is required for calcium responses to bacterial PAMPs (flg22, elf18); core immune process. Reason: BIK1 (with PBL1) is genetically required for MAMP/DAMP-induced calcium elevations triggered by bacteria-derived flg22 and elf18. Core response to bacterial molecules. Supporting Evidence: PMID:25522736 is also required for MAMP/DAMP-induced calcium elevations.
GO:0002237 response to molecule of bacterial origin	IMP PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	ACCEPT	Summary: BIK1 mediates responses to bacterial PAMPs downstream of EFR/FLS2; core immune process. Reason: BIK1 is phosphorylated by the PRR EFR upon perception of bacterial elf18 and is required for elf18/flg22-triggered responses. Core response to bacterial molecules. Supporting Evidence: PMID:29649442 EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1.
GO:0005515 protein binding	IPI PMID:29649442 The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the N...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-EFR and BIK1-WRKY interaction study; uninformative MF although the interactions are biologically central. Reason: Bare GO:0005515 is uninformative. The underlying interactions (BIK1 with the PRR EFR and with nuclear WRKY transcription factors WRKY33/50/51/57) are real and are captured by the kinase MF and the nuclear hormone-regulation BP terms; bare binding is not a core function. Supporting Evidence: PMID:29649442 BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors
GO:1900424 regulation of defense response to bacterium	IMP PMID:25522736 Microbe-associated molecular pattern-induced calcium signali...	ACCEPT	Summary: BIK1 regulates antibacterial defense via control of early MAMP-induced calcium signaling; supported immune function. Reason: BIK1 is required for MAMP/DAMP-induced calcium signaling that initiates antibacterial defense responses, placing it as a regulator of defense response to bacterium. Core-adjacent immune BP. Supporting Evidence: PMID:25522736 is also required for MAMP/DAMP-induced calcium elevations.
GO:0005794 Golgi apparatus	HDA PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerizati...	MARK AS OVER ANNOTATED	Summary: Golgi localization derives only from a global membrane-protein correlation-profiling proteomics survey, not a focused BIK1 study; likely over-annotation. Reason: This HDA call is from a high-throughput protein correlation profiling proteomics study of membrane-protein oligomerization, not a dedicated BIK1 localization study. Golgi residence is not supported by any focused BIK1 work (which establishes plasma membrane, nucleus and endosomes), and likely reflects fractionation/co-migration rather than genuine steady-state Golgi localization. Supporting Evidence: PMID:28887381 Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling.
GO:0002221 pattern recognition receptor signaling pathway	IMP PMID:21862710 Biochemical and genetic requirements for function of the imm...	ACCEPT	Summary: BIK1 is genetically and biochemically required for PAMP-triggered immunity downstream of PRRs; core biological process. Reason: This study established biochemical/genetic requirements for BIK1 in PAMP-triggered immunity (and growth/ethylene signaling). PRR signaling pathway is a core BP for BIK1. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md Central RLCK that relays PTI signaling from PRR–BAK1 complexes
GO:0005739 mitochondrion	ISM GO_REF:0000122	REMOVE	Summary: Mitochondrial localization is a pure sequence-model prediction (AtSubP) with no experimental support and is contradicted by BIK1 biology. Reason: This is an ISM (sequence-model) prediction from AtSubP (GO_REF:0000122) with no experimental backing. BIK1 is N-myristoylated/palmitoylated and anchored to the plasma membrane, with experimentally established nucleus and endosome pools; mitochondrial localization is biologically implausible and contradicted by all focused studies. Demonstrably wrong electronic prediction. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md with no experimental support and is inconsistent with the myristoyl/palmitoyl PM anchor
GO:0005515 protein binding	IPI PMID:20404519 Phosphorylation of receptor-like cytoplasmic kinases by bact...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-FLS2/BAK1 interaction and phosphorylation study; uninformative MF although the interaction is central. Reason: Bare GO:0005515 is uninformative. The interaction (BIK1 with FLS2 and BAK1, with mutual trans-phosphorylation) is genuine and captured by the kinase MF and PRR signaling BP; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md FLS2/BAK1 (PMID:20018686, PMID:20404519, PMID:20413097)
GO:0005515 protein binding	IPI PMID:20413097 Receptor-like cytoplasmic kinases integrate signaling from m...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from study showing BIK1 integrates signaling from multiple immune receptors and is an effector target; uninformative MF. Reason: Bare GO:0005515 is uninformative. The biology (BIK1 associating with multiple PRRs and being targeted by a Pseudomonas effector) is captured by the PRR signaling and defense BP terms; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md BIK1 integrates signaling from multiple immune receptors and is targeted by a Pseudomonas effector
GO:0005515 protein binding	IPI PMID:23431184 BIK1 interacts with PEPRs to mediate ethylene-induced immuni...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-PEPR1 interaction study (ethylene-induced immunity); uninformative MF although the interaction is real. Reason: Bare GO:0005515 is uninformative. The specific interaction (BIK1 with the PEPR1 receptor, mediating Pep1/ethylene-induced immunity) is genuine but is better captured by signaling BP terms; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md PEPR1 (PMID:23431184)
GO:0005515 protein binding	IPI PMID:23818580 Inverse modulation of plant immune and brassinosteroid signa...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-BRI1 interaction study (brassinosteroid signaling); uninformative MF although the interaction is real. Reason: Bare GO:0005515 is uninformative. The specific interaction (BIK1 with the BR receptor BRI1, negatively regulating BR signaling) is genuine but better captured by signaling BP terms; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md Negative regulator of brassinosteroid signaling via BRI1 interaction
GO:0005515 protein binding	IPI PMID:24629339 The FLS2-associated kinase BIK1 directly phosphorylates the ...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from the BIK1-RBOHD study; uninformative as bare MF, but the kinase-substrate relationship is a core part of BIK1 function. Reason: Bare GO:0005515 is uninformative. The underlying interaction (BIK1 binding and directly phosphorylating the NADPH oxidase RBOHD to drive the ROS burst) is a core function captured by the kinase MF and ROS/defense BP terms; bare binding should not stand as a separate functional claim. Supporting Evidence: PMID:24629339 directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation.
GO:0005515 protein binding	IPI PMID:25525792 The calcium-dependent protein kinase CPK28 buffers plant imm...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-CPK28 interaction study (regulation of BIK1 turnover); uninformative MF although the interaction is real. Reason: Bare GO:0005515 is uninformative. The specific interaction (CPK28 binding BIK1 to buffer immunity and regulate BIK1 turnover) is a genuine regulatory relationship better described by the regulation biology; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md CPK28 (PMID:25525792)
GO:0005515 protein binding	IPI PMID:27494702 The Arabidopsis Protein Phosphatase PP2C38 Negatively Regula...	MARK AS OVER ANNOTATED	Summary: Bare protein binding from BIK1-PP2C38 interaction study (negative regulation of BIK1); uninformative MF although the interaction is real. Reason: Bare GO:0005515 is uninformative. The specific interaction (the phosphatase PP2C38 binding and negatively regulating BIK1 activation) is a genuine regulatory relationship; bare binding is not a core function. Supporting Evidence: file:ARATH/BIK1/BIK1-notes.md PP2C38 (PMID:27494702)
GO:0005886 plasma membrane	IDA PMID:16339855 The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinc...	ACCEPT	Summary: Original demonstration that BIK1 is membrane-localized; core location. Reason: The founding BIK1 study showed membrane localization, consistent with its lipid anchoring and role in early pathogen signal transduction. Core location. Supporting Evidence: PMID:16339855 BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response.
GO:0009620 response to fungus	IEP PMID:16339855 The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinc...	KEEP AS NON CORE	Summary: BIK1 transcript is induced by Botrytis infection; expression-based response to fungus, kept as non-core relative to the more specific defense term. Reason: This IEP annotation reflects transcriptional induction of BIK1 by Botrytis cinerea. It is supported but is an expression-based (IEP) inference; the more specific defense response to fungus (IMP) better captures the functional role. Supporting Evidence: PMID:16339855 the Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) gene that is transcriptionally regulated by Botrytis cinerea infection.
GO:0016301 kinase activity	IDA PMID:16339855 The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinc...	MODIFY	Summary: General kinase activity; correct but less specific than the proven protein Ser/Thr kinase activity. Reason: BIK1 is a protein Ser/Thr (dual-specificity) kinase. The generic kinase activity term should be replaced by the specific protein serine/threonine kinase activity term, which is independently and experimentally supported. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:16339855 BIK1 encodes a regulatory protein, specifically a protein kinase, predicted to be specific to Ser/Thr residues
GO:0046777 protein autophosphorylation	IDA PMID:16339855 The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinc...	KEEP AS NON CORE	Summary: BIK1 autophosphorylates on numerous Ser/Thr/Tyr residues; a real activity but a mechanistic/PTM detail rather than the primary biological role. Reason: Autophosphorylation is experimentally well documented (19 in vitro autophosphorylation sites, including phosphotyrosines) and contributes to BIK1 activation, but it is a self-directed catalytic detail; the core function is trans-phosphorylation of downstream immune substrates. Retained as non-core. Supporting Evidence: PMID:24104392 we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites
GO:0050832 defense response to fungus	IMP PMID:16339855 The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinc...	ACCEPT	Summary: bik1 mutants are severely susceptible to necrotrophic fungi, establishing a genuine role in defense response to fungus; core-adjacent immune function. Reason: Loss of BIK1 causes severe susceptibility to the necrotrophic fungi Botrytis cinerea and Alternaria brassicicola, directly demonstrating a required role in antifungal defense. Supporting Evidence: PMID:16339855 Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens

Core Functions

Acts as a dual-specificity (predominantly Ser/Thr) protein kinase that, upon trans-phosphorylation by BAK1 within ligand-activated PRR complexes, directly phosphorylates downstream immune substrates to relay PAMP-triggered immunity signaling.

Molecular Function:

protein serine/threonine kinase activity

Directly Involved In:

pattern recognition receptor signaling pathway response to molecule of bacterial origin

Cellular Locations:

plasma membrane

Supporting Evidence:

PMID:20018686
BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling.
PMID:24104392
we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time.
file:ARATH/BIK1/BIK1-deep-research-falcon.md
connecting PRR activation to early defense outputs such as reactive oxygen species

Directly phosphorylates the NADPH oxidase RBOHD in a calcium-independent manner to drive the apoplastic reactive oxygen species (ROS) burst that underlies antibacterial immunity and stomatal defense.

Molecular Function:

protein serine/threonine kinase activity

Directly Involved In:

defense response to bacterium

Cellular Locations:

plasma membrane

Supporting Evidence:

PMID:24629339
directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation.
file:ARATH/BIK1/BIK1-deep-research-falcon.md
BIK1 directly interacts with and phosphorylates RBOHD following PAMP perception.

Phosphorylates and activates the calcium-permeable channel OSCA1.3 to promote PAMP-induced cytosolic Ca2+ influx and stomatal closure during immune signaling.

Molecular Function:

protein serine/threonine kinase activity

Directly Involved In:

regulation of stomatal movement

Cellular Locations:

plasma membrane

Supporting Evidence:

PMID:32846426
OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling
PMID:25522736
is also required for MAMP/DAMP-induced calcium elevations.

References

file:ARATH/BIK1/BIK1-deep-research-falcon.md

Falcon/Edison deep research report: BIK1

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

GO_REF:0000003

Gene Ontology annotation based on Enzyme Commission mapping

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt

GO_REF:0000116

Automatic Gene Ontology annotation based on Rhea mapping

GO_REF:0000122

AtSubP analysis

DOI:10.1038/s41586-020-2210-3

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

PMID:15610358

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions.

PMID:16339855

The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens.

PMID:20018686

A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity.

PMID:20404519

Phosphorylation of receptor-like cytoplasmic kinases by bacterial flagellin.

PMID:20413097

Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector.

PMID:21862710

Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis.

PMID:23431184

BIK1 interacts with PEPRs to mediate ethylene-induced immunity.

PMID:23818580

Inverse modulation of plant immune and brassinosteroid signaling pathways by the receptor-like cytoplasmic kinase BIK1.

PMID:24104392

Identification and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1.

PMID:24629339

The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity.

PMID:25522736

Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1.

PMID:25525792

The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover.

PMID:26021844

Regulatory role of BOTRYTIS INDUCED KINASE1 in ETHYLENE INSENSITIVE3-dependent gene expression in Arabidopsis.

PMID:27494702

The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.

PMID:28887381

Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling.

PMID:29649442

The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.

PMID:30584105

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

PMID:31803215

BIK1 and ERECTA Play Opposing Roles in Both Leaf and Inflorescence Development in Arabidopsis.

PMID:32404997

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

PMID:32846426

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.

Suggested Experiments

Experiment: Engineer nuclear-excluded (membrane-tethered) and constitutively nuclear BIK1 variants and quantify the ROS burst, Ca2+ influx, stomatal closure, JA/SA levels, and bacterial resistance for each, separating membrane from nuclear outputs.

Hypothesis: BIK1 nuclear relocation is required specifically for hormone-branch immunity but dispensable for the membrane ROS/Ca2+ burst.

Experiment: Systematically combine activation-loop and Tyr phosphosite mutants with in vitro and in vivo phosphorylation assays against RBOHD, OSCA1.3, FLS2/BAK1 and WRKY substrates to map a site-to-substrate code.

Hypothesis: Distinct BIK1 phosphosites gate distinct downstream substrates.

Deep Research

Falcon

(BIK1-deep-research-falcon.md)

Gene Research for GO Annotation Review Falcon citations file

Gene Research for GO Annotation Review

Target

Gene symbol: BIK1
Organism: Arabidopsis thaliana

UniProt Context

=== UNIPROT METADATA ===
UniProt ID: O48814
Entry Name: BIK1_ARATH
Gene Name: BIK1
Locus Tag: At2g39660 {ECO:0000312|Araport:AT2G39660}
Protein Name: Serine/threonine-protein kinase BIK1
EC Number: 2.7.11.1
Organism: Arabidopsis thaliana (Mouse-ear cress)
NCBI Taxonomy ID: 3702
Function: (Microbial infection) Xanthomonas campestris effector AvrAC/XopAC-mediated uridylylation prevents activation by phosphorylation at the same residues, thus affecting immune responses and reducing defense responses toward X.campestris, mediating avrAC/XopAC virulence functions.
Subcellular Location: Cell membrane
Subunit: Interacts with FLS2 (PubMed:20404519, PubMed:20413097). Activation of FLS2 by flagellin (flg22) induces the dissociation of the complex (PubMed:20413097, PubMed:32404997). Interacts with BAK1 (PubMed:20404519). Interacts with the Xanthomonas campestris effector XopAC/AvrAC (PubMed:22504181). Interacts with CPK28 (PubMed:25525792). Interacts with PEPR1 (PubMed:23431184). Interacts with PP2C38 (PubMed:27494702). Interacts with BRI1 (PubMed:23818580). Interacts with RBOHD (PubMed:24629339). Binds to EFR when not phosphorylated at Ser-89 and Thr-90, in the absence of pathogen elicitor; dissociates upon pathogen-associated molecular pattern (PAMP)-triggered activation by EFR-mediated phosphorylation (PubMed:29649442). Interacts directly with and phosphorylates WRKY transcription factors in the nucleus involved in the jasmonic acid (JA) and salicylic acid (SA) regulation (e.g. WRKY33, WRKY50, WRKY51 and WRKY57) to modulate defense hormones during plant immunity (PubMed:29649442). Binds to ATL44/RHA3A and ATL45/RHA3B (PubMed:32404997). Binds to SIK1 to be phosphorylated and stabilized (PubMed:30212650).
======================

Research Objective

Prepare a comprehensive literature-backed research report for GO annotation review of the target gene.

Focus on:

The core biochemical activity of the gene product and any substrate specificity.
The activation or maturation mechanism of the protein, including proteolytic processing if relevant.
The best-supported subcellular localization and complexes where the active protein functions.
The strongest experimentally supported biological processes in the target organism, or in very close orthologs when transfer is justified.
Which annotations appear to reflect core function versus context-specific, pleiotropic, or over-extended roles.
Any evidence bearing on apoptosis, developmental cell death, neuronal roles, inflammatory signaling, pyroptosis, synaptic remodeling, protein processing, and localization to cytoplasm, cytosol, nucleus, or signaling complexes.

Output Requirements

Structure the report as:

Executive summary
Molecular function
Biological process roles
Cellular localization and complexes
Annotation-risk assessment
Key literature

Use citations for all substantive claims.

Gene Research Report for GO Annotation Review: BIK1 (Arabidopsis thaliana; UniProt O48814; At2g39660)

1. Executive summary

BOTRYTIS-INDUCED KINASE 1 (BIK1) encodes a plasma-membrane-associated receptor-like cytoplasmic kinase (RLCK) that acts immediately downstream of multiple pattern-recognition receptors (PRRs) in Arabidopsis innate immunity, where it functions as a signaling hub connecting PRR activation to early defense outputs such as reactive oxygen species (ROS) production, Ca2+ signaling, and stomatal immunity. Core evidence from high-citation primary studies shows that BIK1 is an active serine/threonine kinase that directly phosphorylates the NADPH oxidase RBOHD at defined N-terminal residues (including S39 and S343), enabling the PAMP-triggered oxidative burst. (li2014thefls2associatedkinase pages 1-2, yasuhiro2014directregulationof pages 9-9)

Recent work (2023–2024) shifts part of the “current understanding” from a purely biochemical linear pathway to a spatiotemporally regulated module: (i) BIK1 activation and stability are governed by multiple post-translational modifications (PTMs), including PP2C38-dependent dephosphorylation, S-nitrosylation at Cys80, and a network of E3 ligases that ubiquitinate distinct BIK1 phospho-forms; and (ii) BIK1 positioning in plasma membrane nanodomains is actively organized by myosin XI (XIK), which promotes FLS2–BIK1 complex formation and robust signaling. (daniel2016thearabidopsisprotein pages 1-5, cui2024snitrosylationofa pages 5-6, bai2023bik1proteinhomeostasis pages 1-2, wang2024myosinximediatedbik1 pages 1-2)

For GO curation, the strongest “core” annotations are to protein serine/threonine kinase activity, PRR-triggered immunity / defense response, positive regulation of ROS production, and plasma membrane-associated immune receptor complexes. In contrast, claims about broad developmental roles, generalized “cell death” regulation, or animal-specific pathways (apoptosis, pyroptosis, neuronal/synaptic functions, inflammatory signaling) are not supported by the core mechanistic evidence base presented here and should be treated as out of scope or as indirect/pleiotropic unless backed by targeted Arabidopsis experiments. (yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)

2. Molecular function

2.1 Key concepts/definitions (current understanding)

Receptor-like cytoplasmic kinases (RLCKs) are non-transmembrane kinases that couple cell-surface receptors (PRRs) to intracellular signaling. In this framework, BIK1 functions as a PRR-associated kinase, rapidly phosphorylated upon ligand perception and then released from PRR complexes to phosphorylate downstream effectors. (yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)

2.2 Core biochemical activity and substrate specificity

Kinase type: Experimental data support BIK1 as an active serine/threonine protein kinase. (yasuhiro2014directregulationof pages 1-2, li2014thefls2associatedkinase pages 1-2)

Direct substrate: Two landmark 2014 studies provide direct evidence that BIK1 phosphorylates the NADPH oxidase RBOHD:
- BIK1 directly phosphorylates RBOHD (calcium independent) at key residues including S39 and S343, enhancing ROS production. (li2014thefls2associatedkinase pages 1-2)
- Complementary phosphoproteomic/targeted MS evidence identifies multiple BIK1-dependent sites on RBOHD including S39, S339, S343 (and additional sites such as T123, S140, S347 in vitro), and links these sites to PAMP-induced phosphorylation within minutes after elicitation. (yasuhiro2014directregulationof pages 9-9, yasuhiro2014directregulationof pages 6-7)

Quantitative/statistical notes: In the Molecular Cell study, phosphorylation-site identification and dynamics were supported by targeted mass spectrometry (SRM) and functional testing of phospho-dead/mimic mutants, and ROS burst assays were quantified over tens of minutes with replicated leaf-disc measurements and strong statistical thresholds (e.g., ANOVA/Tukey, p < 0.001). (yasuhiro2014directregulationof pages 9-9)

2.3 Activation and inactivation mechanisms (maturation/processing)

PRR-complex phosphorylation and release: BIK1 is described as constitutively associated with PRRs (e.g., FLS2 and EFR) and becomes rapidly phosphorylated and released upon PAMP perception. (yasuhiro2014directregulationof pages 1-2)

Negative regulation by phosphatase PP2C38: PP2C38 is identified as a negative regulator that dynamically associates with BIK1 and PRRs (FLS2/EFR) and impairs PAMP-induced BIK1 phosphorylation and BIK1-mediated RBOHD phosphorylation; upon PAMP perception PP2C38 is phosphorylated and dissociates, enabling full BIK1 activation. (daniel2016thearabidopsisprotein pages 1-5)

Redox activation via S-nitrosylation (2024): A 2024 Science Advances study reports that P/MAMP perception triggers a nitrosative burst leading to S-nitrosylation of BIK1 at Cys80, which promotes BIK1 phosphorylation/activation and stabilization and increases physical interaction with RBOHD, boosting ROS formation. The study uses targeted mutagenesis (e.g., BIK1C80A) and shows reduced ROS kinetics (measured over 60 minutes following flg22 treatment) and compromised resistance to a nonpathogenic bacterial strain in complementation assays. (cui2024snitrosylationofa pages 5-6)

Ubiquitin-mediated control of BIK1 pools (2023–2024): Recent evidence and synthesis describe a multi-E3 network that differentially ubiquitinates BIK1 depending on its phosphorylation state:
- PUB25/26: preferentially ubiquitinate hypo-phosphorylated BIK1 for degradation, acting as negative regulators of immune signaling. (bai2023bik1proteinhomeostasis pages 1-2, fu2024themultifacetedubiquitination pages 6-7)
- RGLG1/2 (Nature Communications 2023): preferentially associate with hypo-phosphorylated BIK1, promote BIK1 accumulation and BAK1–BIK1 association, and antagonize PUB25-mediated degradation; a reported phospho-mimic (BIK1S236D/T237D) shows resistance to PUB25 ubiquitination. (bai2023bik1proteinhomeostasis pages 1-2, bai2023bik1proteinhomeostasis pages 8-9)
- RHA3A/B: mediate PAMP-induced monoubiquitination of activated/hyperphosphorylated BIK1 (observed as an ~8 kDa shift consistent with monoubiquitination), promoting release from FLS2–BAK1 and internalization to endocytic vesicles to enable signaling. (fu2024themultifacetedubiquitination pages 6-7)
- PUB4: described as having dual roles, promoting degradation of non-activated BIK1 at rest while supporting accumulation/associations of activated BIK1 after PAMP perception. (bai2023bik1proteinhomeostasis pages 1-2, fu2024themultifacetedubiquitination pages 6-7)

Site-level ubiquitination (from 2024 synthesis): BIK1 has many lysines, and mutational/MS analyses implicate multiple ubiquitination sites (e.g., K31, K41, K95, K170, K186, K286, K337, K358, K366 among others), with multi-lysine mutants (e.g., “9KR”) substantially reducing monoubiquitination and dissociation from PRR complexes. (fu2024themultifacetedubiquitination pages 6-7, fu2024themultifacetedubiquitination pages 7-9)

Proteolytic targeting by pathogen effectors: In the immune context, bacterial type-III effectors can proteolytically cleave RLCKs; a recent synthesis notes AvrPphB cleavage of BIK1 in plant–pathogen interactions. This is not a normal maturation step for the host protein but a pathogen virulence strategy. (fu2024themultifacetedubiquitination pages 2-4, daniel2016thearabidopsisprotein pages 1-5)

3. Biological process roles

3.1 Best-supported processes in Arabidopsis (direct evidence)

Pattern-triggered immunity (PTI) / innate immune signaling: BIK1 is repeatedly described as a PRR-associated kinase acting downstream of multiple PRRs (FLS2, EFR; also CERK1, PEPR1), linking PAMP perception to downstream defense signaling. (daniel2016thearabidopsisprotein pages 1-5, yasuhiro2014directregulationof pages 1-2)

Positive regulation of the ROS burst: Direct phosphorylation of RBOHD by BIK1 and requirement of BIK1-dependent RBOHD phosphosites for PAMP-triggered ROS bursts are central functional outputs, supported by both biochemical and genetic evidence (RBOHD phosphosite mutants, bik1-related mutants). (yasuhiro2014directregulationof pages 9-9, li2014thefls2associatedkinase pages 1-2)

Stomatal immunity: BIK1-dependent signaling through RBOHD is linked to stomatal movement that restricts bacterial entry, with impairment when BIK1 function (or its RBOHD phosphorylation) is compromised. (li2014thefls2associatedkinase pages 1-2, daniel2016thearabidopsisprotein pages 1-5)

Early Ca2+ signaling: BIK1 (with PBL1) is required for MAMP/DAMP-induced calcium elevations, positioning BIK1 in early signaling outputs beyond ROS. (li2014thefls2associatedkinase pages 1-2)

3.2 Recent developments (2023–2024) refining process interpretation

Immune homeostasis as a regulated “BIK1 pool” problem: 2023–2024 work emphasizes that BIK1 is not simply “on/off,” but rather immune amplitude is tuned by the relative abundance of hypophosphorylated vs activated BIK1 and by distinct ubiquitin ligase modules (RGLG1/2 vs PUB25/26 vs RHA3A/B vs PUB4). This reframes some GO biological-process interpretation toward “regulation of protein stability/turnover in immune signaling” rather than only “defense response.” (bai2023bik1proteinhomeostasis pages 1-2, fu2024themultifacetedubiquitination pages 6-7)

Redox–ROS coupling: The 2024 S-nitrosylation mechanism provides a mechanistic bridge between a rapid nitrosative burst and the canonical ROS burst by directly modulating BIK1 activation and BIK1–RBOHD interaction, which is relevant to annotating regulation of ROS biosynthetic process and redox signaling. (cui2024snitrosylationofa pages 5-6)

Spatial organization of signaling (nanodomain recruitment): The 2024 PNAS study adds a cellular-systems layer: myosin XIK recruits/stabilizes BIK1 in FLS2-containing nanodomains, facilitating receptor complex assembly and full activation of BIK1-dependent responses. This supports cellular-component/biological-process annotations related to plasma membrane microdomains and regulation of receptor complex assembly. (wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 42cf9646)

3.3 Current applications and real-world implementations

BIK1 is widely treated as a central node for engineering or breeding disease resistance because it integrates multiple PRR pathways and controls early defense outputs (ROS, Ca2+, stomatal closure). In practical terms, “applications” often focus on manipulating upstream PRRs/co-receptors, E3 ligases controlling BIK1 abundance, or signaling amplitude to enhance resistance while avoiding autoimmunity; recent work on ubiquitin homeostasis modules provides candidate intervention points (e.g., adjusting PUB25/26 or RGLG1/2 balance) to tune defense strength. (bai2023bik1proteinhomeostasis pages 1-2, fu2024themultifacetedubiquitination pages 4-6)

4. Cellular localization and complexes

4.1 Best-supported subcellular localization

Plasma membrane association: BIK1 is consistently described as plasma-membrane-associated and PRR-complex associated. (yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)

Nanodomain localization (2024): Imaging evidence supports enrichment of BIK1 in FLS2-containing plasma membrane nanodomains, promoted by myosin XIK. (wang2024myosinximediatedbik1 media 42cf9646, wang2024myosinximediatedbik1 pages 1-2)

Endocytic trafficking: RHA3A/B-dependent monoubiquitination is described as promoting internalization of BIK1 from the plasma membrane to endocytic vesicles for signaling activation (distinguished from FLS2 internalization for attenuation). (fu2024themultifacetedubiquitination pages 6-7)

Nuclear localization (contextual): A 2024 synthesis notes that BIK1 can localize to the nucleus and phosphorylate WRKY transcription factors, but also states uncertainty about how BIK1 translocates from plasma membrane to nucleus. This suggests nuclear localization should be curated cautiously unless supported by direct primary evidence in Arabidopsis tissues/conditions. (fu2024themultifacetedubiquitination pages 2-4)

4.2 Complexes and direct interactors

PRR-associated complexes: BIK1 is described as constitutively associating with FLS2 and EFR (and with BAK1 in those receptor complexes), and also associating with CERK1 (chitin signaling) and PEPR1 pathways. (yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)

RBOHD complex: BIK1 directly interacts with and phosphorylates RBOHD following PAMP perception. (yasuhiro2014directregulationof pages 1-2, li2014thefls2associatedkinase pages 1-2)

Myosin XI (XIK)–organized complexes (2024): XIK–BIK1 interaction and XIK-dependent confinement in nanodomains is proposed to stabilize immune receptor complex assembly. This provides a mechanistic basis for annotating BIK1 in a spatially organized plasma membrane signaling complex. (wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 97a839e6)

Supporting figure evidence: The PNAS 2024 figure panels show BIK1 colocalization with FLS2 at the plasma membrane and BIK1 recruitment to REM1.3-marked nanodomains, both reduced by myosin inhibition, plus a model summarizing myosin XI roles in BIK1 delivery and nanodomain immobilization. (wang2024myosinximediatedbik1 media 42cf9646, wang2024myosinximediatedbik1 media 7765405b, wang2024myosinximediatedbik1 media 97a839e6)

5. Annotation-risk assessment (core vs context-specific; over-extension risks)

5.1 High-confidence “core function” annotations (recommended emphasis)

Protein serine/threonine kinase activity (BIK1 is an active RLCK). (yasuhiro2014directregulationof pages 1-2, li2014thefls2associatedkinase pages 1-2)
Direct phosphorylation of RBOHD (defined sites including S39/S343; strong mechanistic linkage to ROS). (li2014thefls2associatedkinase pages 1-2, yasuhiro2014directregulationof pages 9-9)
Positive regulation of PAMP-triggered ROS burst / PTI and associated immune outputs such as stomatal immunity and early Ca2+ signaling. (li2014thefls2associatedkinase pages 1-2, daniel2016thearabidopsisprotein pages 1-5)
Cellular component: plasma membrane / PRR-associated complex; plus nanodomain association supported by 2024 imaging. (yasuhiro2014directregulationof pages 1-2, wang2024myosinximediatedbik1 media 42cf9646)

5.2 Context-specific regulatory annotations (valid but should be framed as regulation of BIK1 in immunity)

Negative regulation via phosphatase PP2C38 (dynamic association/dissociation). (daniel2016thearabidopsisprotein pages 1-5)
Redox regulation via S-nitrosylation at Cys80 (PTI-specific mechanism with clear phenotypes). (cui2024snitrosylationofa pages 5-6)
Ubiquitination-based homeostasis (distinct E3 ligases act on distinct BIK1 pools; immune amplitude control). (bai2023bik1proteinhomeostasis pages 1-2, fu2024themultifacetedubiquitination pages 6-7)
Protein localization to nanodomains and receptor complex assembly regulated by myosin XIK (a 2024 mechanistic addition). (wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 97a839e6)

5.3 Over-extension risks / terms to avoid without additional direct evidence

Apoptosis, pyroptosis, mammalian inflammatory signaling, neuronal/synaptic remodeling: These are animal-centric pathways and are not discussed in the primary BIK1 mechanistic literature examined here; thus annotations implying such processes are not justified for Arabidopsis BIK1 based on this evidence set. (yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)
Generic “programmed cell death” claims: Not supported by the cited mechanistic evidence; any such annotation would require direct Arabidopsis experiments showing BIK1-dependent cell death phenotypes under defined triggers, distinct from PTI outputs. (yasuhiro2014directregulationof pages 1-2)
Proteolytic processing as maturation: Evidence for cleavage is presented as pathogen effector-mediated targeting, not host maturation/processing; thus avoid annotations implying normal proteolytic activation. (fu2024themultifacetedubiquitination pages 2-4)
Nuclear localization as a constitutive cellular component: A 2024 synthesis mentions nuclear activity but also notes uncertainty on the mechanism; nuclear CC annotations should be conservative and evidence-driven (prefer primary nuclear localization data). (fu2024themultifacetedubiquitination pages 2-4)

6. Key literature (priority to 2023–2024; includes URLs and publication dates)

High-priority recent primary research (2023–2024)

Bai et al. (Aug 2023). BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling. Nature Communications 14. https://doi.org/10.1038/s41467-023-40364-0 (bai2023bik1proteinhomeostasis pages 1-2, bai2023bik1proteinhomeostasis pages 8-9)
Wang et al. (Jun 2024). Myosin XI-mediated BIK1 recruitment to nanodomains facilitates FLS2–BIK1 complex formation during innate immunity in Arabidopsis. PNAS 121(25). https://doi.org/10.1073/pnas.2312415121 (wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 42cf9646)
Cui et al. (Mar 2024). S-nitrosylation of a receptor-like cytoplasmic kinase regulates plant immunity. Science Advances 10(11). https://doi.org/10.1126/sciadv.adk3126 (cui2024snitrosylationofa pages 5-6)
Fu et al. (Nov 2024). The Multifaceted Ubiquitination of BIK1 During Plant Immunity in Arabidopsis thaliana. International Journal of Molecular Sciences 25(22):12187. https://doi.org/10.3390/ijms252212187 (fu2024themultifacetedubiquitination pages 6-7)

Foundational mechanistic literature

Kadota et al. (Apr 2014). Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Molecular Cell 54(1):43–55. https://doi.org/10.1016/j.molcel.2014.02.021 (yasuhiro2014directregulationof pages 9-9, yasuhiro2014directregulationof pages 6-7)
Li et al. (Mar 2014). The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host & Microbe 15(3):329–338. https://doi.org/10.1016/j.chom.2014.02.009 (li2014thefls2associatedkinase pages 1-2)
Couto et al. (Aug 2016). The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1. PLoS Pathogens 12(8):e1005811. https://doi.org/10.1371/journal.ppat.1005811 (daniel2016thearabidopsisprotein pages 1-5)

GO-oriented evidence summary table

The following table consolidates candidate GO terms with the strongest supporting evidence, including residues/mutants and primary references.

GO aspect	Candidate GO term (plain text)	Key evidence/assay (1 phrase)	Key residues/mutants (if any)	Primary reference (authors/year/journal) with URL	Citation ID
MF	protein serine/threonine kinase activity	In vitro kinase assays and phosphosite mapping support BIK1 as an active RLCK kinase	Kinase-dead BIK1K105E noted in regulatory studies; ATP-pocket K105/K106 important	Kadota et al., 2014, Molecular Cell. https://doi.org/10.1016/j.molcel.2014.02.021 ; Li et al., 2014, Cell Host & Microbe. https://doi.org/10.1016/j.chom.2014.02.009	(yasuhiro2014directregulationof pages 1-2, li2014thefls2associatedkinase pages 1-2)
MF	protein kinase activity acting on RBOHD	Direct phosphorylation of RBOHD by BIK1 shown by in vitro kinase/SRM and in vivo phosphosite analysis	RBOHD S39, T123, S140, S339, S343, S347; phospho-dead/mimic RBOHD mutants tested	Kadota et al., 2014, Molecular Cell. https://doi.org/10.1016/j.molcel.2014.02.021 ; Li et al., 2014, Cell Host & Microbe. https://doi.org/10.1016/j.chom.2014.02.009	(yasuhiro2014directregulationof pages 9-9, yasuhiro2014directregulationof pages 6-7, li2014thefls2associatedkinase pages 1-2)
BP	positive regulation of reactive oxygen species production	flg22/PAMP-induced ROS burst reduced in bik1 mutants and RBOHD phosphosite mutants	bik1; bik1 pbl1; RBOHD S39A/S339A/S343A and phosphomimics	Kadota et al., 2014, Molecular Cell. https://doi.org/10.1016/j.molcel.2014.02.021 ; Li et al., 2014, Cell Host & Microbe. https://doi.org/10.1016/j.chom.2014.02.009	(yasuhiro2014directregulationof pages 9-9, li2014thefls2associatedkinase pages 1-2, yasuhiro2014directregulationof pages 6-7)
BP	regulation of calcium ion influx during immune signaling	aequorin-based calcium assays show BIK1/PBL1 required for MAMP-induced calcium elevations	bik1; pbl1/cce5	Li et al., 2014, Cell Host & Microbe. https://doi.org/10.1016/j.chom.2014.02.009 ; Ranf et al., 2014, BMC Plant Biology. https://doi.org/10.1186/s12870-014-0374-4	(li2014thefls2associatedkinase pages 1-2, fu2024themultifacetedubiquitination pages 2-4)
CC	plasma membrane PRR complex	Co-IP/association studies show BIK1 constitutively associates with FLS2/EFR/BAK1 and dissociates after PAMP perception	FLS2, EFR, BAK1 complexes; CERK1 also associated	Kadota et al., 2014, Molecular Cell. https://doi.org/10.1016/j.molcel.2014.02.021 ; Couto et al., 2016, PLoS Pathogens. https://doi.org/10.1371/journal.ppat.1005811	(yasuhiro2014directregulationof pages 1-2, daniel2016thearabidopsisprotein pages 1-5)
BP	negative regulation of BIK1 signaling by dephosphorylation	PP2C38 association/dissociation and reduced BIK1-dependent RBOHD phosphorylation	PP2C38 Ser77 phosphorylation; pp2c38 functional tests	Couto et al., 2016, PLoS Pathogens. https://doi.org/10.1371/journal.ppat.1005811	(daniel2016thearabidopsisprotein pages 1-5)
BP	activation of BIK1 by S-nitrosylation	GSNO/flg22 assays show Cys80 S-nitrosylation promotes BIK1 phosphorylation, stability, and RBOHD interaction	BIK1C80A; BIK1CA	Cui et al., 2024, Science Advances. https://doi.org/10.1126/sciadv.adk3126	(cui2024snitrosylationofa pages 5-6)
BP	regulation of BIK1 protein stability by ubiquitination	Genetic/biochemical analyses define opposing E3 ligases controlling hypo- vs activated BIK1 pools	PUB25/26; RGLG1/2; RHA3A/B; PUB4; BIK1S236D/T237D resistant to PUB25; BIK19KR	Bai et al., 2023, Nature Communications. https://doi.org/10.1038/s41467-023-40364-0 ; Fu et al., 2024, International Journal of Molecular Sciences. https://doi.org/10.3390/ijms252212187	(bai2023bik1proteinhomeostasis pages 1-2, bai2023bik1proteinhomeostasis pages 8-9, fu2024themultifacetedubiquitination pages 4-6, fu2024themultifacetedubiquitination pages 6-7, fu2024themultifacetedubiquitination pages 9-10)
CC	plasma membrane nanodomain	Imaging/single-particle tracking show myosin XIK recruits BIK1 into FLS2-containing nanodomains	myosin XIK; BDM inhibition; REM1.3-marked nanodomains	Wang et al., 2024, PNAS. https://doi.org/10.1073/pnas.2312415121	(wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 42cf9646)
BP	protein localization to plasma membrane nanodomain facilitating immune complex assembly	Myosin-dependent recruitment stabilizes FLS2–BIK1 complex formation and supports full BIK1-dependent defense output	myosin XIK; BDM-sensitive colocalization	Wang et al., 2024, PNAS. https://doi.org/10.1073/pnas.2312415121	(wang2024myosinximediatedbik1 pages 1-2, wang2024myosinximediatedbik1 media 42cf9646, wang2024myosinximediatedbik1 media 97a839e6)
BP/CC	endocytosis/internalization following monoubiquitination	RHA3A/B-mediated monoubiquitination promotes release from FLS2-BAK1 and internalization to endocytic vesicles	BIK1 9KR; ubiquitin sites including K31, K41, K95, K170, K186, K286, K337, K358, K366	Fu et al., 2024, International Journal of Molecular Sciences. https://doi.org/10.3390/ijms252212187	(fu2024themultifacetedubiquitination pages 6-7, fu2024themultifacetedubiquitination pages 7-9)

Table: This table compiles high-confidence GO-relevant statements for Arabidopsis thaliana BIK1, emphasizing experimentally supported molecular function, biological process, and cellular component assignments. It is useful for annotation review because it links each candidate term to specific assays, residues or mutants, primary literature, and citable context IDs.

References

(li2014thefls2associatedkinase pages 1-2): Lei Li, Meng Li, Liping Yu, Zhaoyang Zhou, Xiangxiu Liang, Zixu Liu, Gaihong Cai, Liyan Gao, Xiaojuan Zhang, Yingchun Wang, She Chen, and Jian-Min Zhou. The fls2-associated kinase bik1 directly phosphorylates the nadph oxidase rbohd to control plant immunity. Cell host & microbe, 15 3:329-38, Mar 2014. URL: https://doi.org/10.1016/j.chom.2014.02.009, doi:10.1016/j.chom.2014.02.009. This article has 964 citations and is from a highest quality peer-reviewed journal.
(yasuhiro2014directregulationof pages 9-9): Yasuhiro Kadota, Jan Sklenar, Paul Derbyshire, Lena Stransfeld, Shuta Asai, Vardis Ntoukakis, Jonathan DG Jones, Ken Shirasu, Frank Menke, Alexandra Jones, and Cyril Zipfel. Direct regulation of the nadph oxidase rbohd by the prr-associated kinase bik1 during plant immunity. Molecular cell, 54 1:43-55, Apr 2014. URL: https://doi.org/10.1016/j.molcel.2014.02.021, doi:10.1016/j.molcel.2014.02.021. This article has 1161 citations and is from a highest quality peer-reviewed journal.
(daniel2016thearabidopsisprotein pages 1-5): Daniel Couto, Roda Niebergall, Xiangxiu Liang, Christoph A Bücherl, Jan Sklenar, Alberto P Macho, Vardis Ntoukakis, Paul Derbyshire, Denise Altenbach, Dan Maclean, Silke Robatzek, Joachim Uhrig, Frank Menke, Jian-Min Zhou, and Cyril Zipfel. The arabidopsis protein phosphatase pp2c38 negatively regulates the central immune kinase bik1. PLoS Pathogens, Aug 2016. URL: https://doi.org/10.1371/journal.ppat.1005811, doi:10.1371/journal.ppat.1005811. This article has 160 citations and is from a highest quality peer-reviewed journal.
(cui2024snitrosylationofa pages 5-6): Beimi Cui, Qiaona Pan, Wenqiang Cui, Yiqin Wang, Verity I. P. Loake, Shuguang Yuan, Fengquan Liu, and Gary J. Loake. S-nitrosylation of a receptor-like cytoplasmic kinase regulates plant immunity. Science Advances, Mar 2024. URL: https://doi.org/10.1126/sciadv.adk3126, doi:10.1126/sciadv.adk3126. This article has 25 citations and is from a highest quality peer-reviewed journal.
(bai2023bik1proteinhomeostasis pages 1-2): Jiaojiao Bai, Yuanyuan Zhou, Jianhang Sun, Kexin Chen, Yufang Han, Ranran Wang, Yanmin Zou, Mingshuo Du, and Dongping Lu. Bik1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling. Nature Communications, Aug 2023. URL: https://doi.org/10.1038/s41467-023-40364-0, doi:10.1038/s41467-023-40364-0. This article has 33 citations and is from a highest quality peer-reviewed journal.
(wang2024myosinximediatedbik1 pages 1-2): Bingxiao Wang, Zhaoyang Zhou, Jian-Min Zhou, and Jiejie Li. Myosin xi-mediated bik1 recruitment to nanodomains facilitates fls2–bik1 complex formation during innate immunity in arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, Jun 2024. URL: https://doi.org/10.1073/pnas.2312415121, doi:10.1073/pnas.2312415121. This article has 14 citations and is from a highest quality peer-reviewed journal.
(yasuhiro2014directregulationof pages 1-2): Yasuhiro Kadota, Jan Sklenar, Paul Derbyshire, Lena Stransfeld, Shuta Asai, Vardis Ntoukakis, Jonathan DG Jones, Ken Shirasu, Frank Menke, Alexandra Jones, and Cyril Zipfel. Direct regulation of the nadph oxidase rbohd by the prr-associated kinase bik1 during plant immunity. Molecular cell, 54 1:43-55, Apr 2014. URL: https://doi.org/10.1016/j.molcel.2014.02.021, doi:10.1016/j.molcel.2014.02.021. This article has 1161 citations and is from a highest quality peer-reviewed journal.
(yasuhiro2014directregulationof pages 6-7): Yasuhiro Kadota, Jan Sklenar, Paul Derbyshire, Lena Stransfeld, Shuta Asai, Vardis Ntoukakis, Jonathan DG Jones, Ken Shirasu, Frank Menke, Alexandra Jones, and Cyril Zipfel. Direct regulation of the nadph oxidase rbohd by the prr-associated kinase bik1 during plant immunity. Molecular cell, 54 1:43-55, Apr 2014. URL: https://doi.org/10.1016/j.molcel.2014.02.021, doi:10.1016/j.molcel.2014.02.021. This article has 1161 citations and is from a highest quality peer-reviewed journal.
(fu2024themultifacetedubiquitination pages 6-7): Junhong Fu, Huihui Wang, Yuling Chen, Chunguang Zhang, and Yanmin Zou. The multifaceted ubiquitination of bik1 during plant immunity in arabidopsis thaliana. International Journal of Molecular Sciences, 25:12187, Nov 2024. URL: https://doi.org/10.3390/ijms252212187, doi:10.3390/ijms252212187. This article has 4 citations.
(bai2023bik1proteinhomeostasis pages 8-9): Jiaojiao Bai, Yuanyuan Zhou, Jianhang Sun, Kexin Chen, Yufang Han, Ranran Wang, Yanmin Zou, Mingshuo Du, and Dongping Lu. Bik1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling. Nature Communications, Aug 2023. URL: https://doi.org/10.1038/s41467-023-40364-0, doi:10.1038/s41467-023-40364-0. This article has 33 citations and is from a highest quality peer-reviewed journal.
(fu2024themultifacetedubiquitination pages 7-9): Junhong Fu, Huihui Wang, Yuling Chen, Chunguang Zhang, and Yanmin Zou. The multifaceted ubiquitination of bik1 during plant immunity in arabidopsis thaliana. International Journal of Molecular Sciences, 25:12187, Nov 2024. URL: https://doi.org/10.3390/ijms252212187, doi:10.3390/ijms252212187. This article has 4 citations.
(fu2024themultifacetedubiquitination pages 2-4): Junhong Fu, Huihui Wang, Yuling Chen, Chunguang Zhang, and Yanmin Zou. The multifaceted ubiquitination of bik1 during plant immunity in arabidopsis thaliana. International Journal of Molecular Sciences, 25:12187, Nov 2024. URL: https://doi.org/10.3390/ijms252212187, doi:10.3390/ijms252212187. This article has 4 citations.
(wang2024myosinximediatedbik1 media 42cf9646): Bingxiao Wang, Zhaoyang Zhou, Jian-Min Zhou, and Jiejie Li. Myosin xi-mediated bik1 recruitment to nanodomains facilitates fls2–bik1 complex formation during innate immunity in arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, Jun 2024. URL: https://doi.org/10.1073/pnas.2312415121, doi:10.1073/pnas.2312415121. This article has 14 citations and is from a highest quality peer-reviewed journal.
(fu2024themultifacetedubiquitination pages 4-6): Junhong Fu, Huihui Wang, Yuling Chen, Chunguang Zhang, and Yanmin Zou. The multifaceted ubiquitination of bik1 during plant immunity in arabidopsis thaliana. International Journal of Molecular Sciences, 25:12187, Nov 2024. URL: https://doi.org/10.3390/ijms252212187, doi:10.3390/ijms252212187. This article has 4 citations.
(wang2024myosinximediatedbik1 media 97a839e6): Bingxiao Wang, Zhaoyang Zhou, Jian-Min Zhou, and Jiejie Li. Myosin xi-mediated bik1 recruitment to nanodomains facilitates fls2–bik1 complex formation during innate immunity in arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, Jun 2024. URL: https://doi.org/10.1073/pnas.2312415121, doi:10.1073/pnas.2312415121. This article has 14 citations and is from a highest quality peer-reviewed journal.
(wang2024myosinximediatedbik1 media 7765405b): Bingxiao Wang, Zhaoyang Zhou, Jian-Min Zhou, and Jiejie Li. Myosin xi-mediated bik1 recruitment to nanodomains facilitates fls2–bik1 complex formation during innate immunity in arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, Jun 2024. URL: https://doi.org/10.1073/pnas.2312415121, doi:10.1073/pnas.2312415121. This article has 14 citations and is from a highest quality peer-reviewed journal.
(fu2024themultifacetedubiquitination pages 9-10): Junhong Fu, Huihui Wang, Yuling Chen, Chunguang Zhang, and Yanmin Zou. The multifaceted ubiquitination of bik1 during plant immunity in arabidopsis thaliana. International Journal of Molecular Sciences, 25:12187, Nov 2024. URL: https://doi.org/10.3390/ijms252212187, doi:10.3390/ijms252212187. This article has 4 citations.

📚 Additional Documentation

Notes

(BIK1-notes.md)

BIK1 (BOTRYTIS-INDUCED KINASE 1, AT2G39660, UniProt O48814) — research notes

Research journal for the GO annotation review of Arabidopsis thaliana BIK1. Provenance recorded inline as [PMID:xxxx "verbatim quote"].

Identity and architecture

BIK1 is a receptor-like cytoplasmic kinase (RLCK, subfamily VII) — a genuine, catalytically active Ser/Thr (dual-specificity) protein kinase. UniProt: "RecName: Full=Serine/threonine-protein kinase BIK1"; EC=2.7.11.1; 395 AA; Protein kinase domain 67-356; ATP-binding Lys-105; active-site (proton acceptor) Asp-202.
Lipid-anchored at the plasma membrane: N-myristoyl Gly-2 and S-palmitoyl Cys-4. Gly-2 mutations mislocalize the protein [PMID:26021844 myristoylation at Gly-2; G->S "Drastic reduction of plasma membrane localization and strong increase of cytoplasmic localization."].
Dual-specificity kinase: PMID:24104392. Catalytic activity (EC 2.7.11.1) experimentally established [PMID:24104392, PMID:32846426].

Core function: immune signaling hub downstream of cell-surface PRRs

Central RLCK that relays PTI signaling from PRR–BAK1 complexes: PMID:20018686, and PMID:20018686.
Trans-phosphorylation order: PMID:20018686.
BIK1 integrates signaling from multiple immune receptors and is targeted by a Pseudomonas effector (title PMID:20413097). Phosphorylated at Ser-236 by FLS2-complex.
UniProt FUNCTION: "Plays a central role in immune responses ... Involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) signaling, including calcium signaling, and defense responses downstream of FLS2."

Core function: phosphorylation of RBOHD and ROS regulation

BIK1 directly phosphorylates the NADPH oxidase RBOHD to drive the ROS burst: PMID:24629339, and PMID:24629339.
Specific RBOHD sites: PMID:29649442.

Core function: phosphorylation of Ca2+ channel OSCA1.3 / stomatal immunity

PMID:32846426, and PMID:32846426.
Phosphorylation depends on kinase activity: PMID:32846426.
BIK1/PBL1 required for MAMP/DAMP-induced calcium elevations: PMID:25522736, and PMID:25522736.

Defense response / hormone regulation

Original identification: required for resistance to necrotrophs, negative regulator of basal resistance to Pst, modulates SA: PMID:16339855, and PMID:16339855. The IEP "response to fungus" annotation derives from: PMID:16339855.
JA/SA regulation via nuclear WRKY phosphorylation: PMID:29649442, and PMID:29649442.

Subcellular localization assessment

Plasma membrane is the principal, well-supported location (lipid anchor; multiple focused studies): UniProt SUBCELLULAR LOCATION "Cell membrane ... Lipid-anchor". Supported by PMID:16339855, PMID:26021844, PMID:29649442, PMID:32404997, and the OSCA paper EXP annotation PMID:32846426.
Nucleus is supported by a focused study with a mechanism (activation-dependent relocation): PMID:29649442; UniProt Note "Linked to the plasma membrane when inactivated, but moves to the nucleus upon pathogen-mediated activation by phosphorylation."
Endosome / endosome membrane / endomembrane supported by ligand-induced internalization study [PMID:32404997 monoubiquitination and internalization "into endocytic compartments"]. UniProt "Endosome membrane".
Nucleolus (GO:0005730), cytoplasm (GO:0005737, HDA), Golgi (GO:0005794, HDA), mitochondrion (GO:0005739, ISM) are from generic high-throughput / predictive sources, NOT focused BIK1 studies:
Nucleolus + cytoplasm + nucleus HDA all come from a high-throughput GFP-ORF transient-expression localization screen: PMID:15610358. This GFP-ORF fusion overexpression assay bypasses native N-myristoylation/palmitoylation membrane targeting and is unreliable for an endogenously membrane-anchored kinase. Nucleolar localization has never been substantiated by any focused BIK1 study.
Golgi HDA is from a global membrane-protein correlation-profiling proteomics survey: PMID:28887381 — not a localization claim specific to BIK1 biology.
Mitochondrion is ISM (sequence-model prediction, GO_REF:0000122 "AtSubP analysis") with no experimental support and is inconsistent with the myristoyl/palmitoyl PM anchor.

Developmental / growth-defense tradeoff (non-core)

bik1 has pleiotropic growth phenotypes: PMID:16339855. UniProt DISRUPTION PHENOTYPE: "Altered growth traits, early flowering, weak stems, small siliques and reduced fertility."
Opposing role with ERECTA in development; BIK1 phosphorylates ER: PMID:31803215. (Basis for one of the protein-binding IPI annotations.)
Negative regulator of brassinosteroid signaling via BRI1 interaction [PMID:23818580 title]; ethylene signaling via EIN3 destabilization PMID:26021844.

Protein-binding (IPI) annotations

BIK1 has many GO:0005515 "protein binding" IPI annotations citing interaction studies. The specific, informative relationships captured by these papers are: FLS2/BAK1 (PMID:20018686, PMID:20404519, PMID:20413097), PEPR1 (PMID:23431184), BRI1 (PMID:23818580), RBOHD (PMID:24629339), CPK28 (PMID:25525792), PP2C38 (PMID:27494702), EFR + WRKY TFs (PMID:29649442), ERECTA (PMID:31803215), the AvrAC/XopAC complex (DOI:10.1038/s41586-020-2210-3 references). Bare "protein binding" is uninformative and should not be treated as a core molecular function; the relevant kinase-substrate / receptor-association relationships are captured in the MF kinase term and the BP signaling terms. Per project guidelines, bare protein binding is demoted (MARK_AS_OVER_ANNOTATED / KEEP_AS_NON_CORE), not removed (these are real IPI experimental annotations).

Summary of curation decisions

Kinase activity terms (GO:0004672, GO:0004674, GO:0106310, GO:0016301) and ATP binding (GO:0005524): ACCEPT (active kinase, experimentally proven; specific Ser/Thr kinase activity is core).
protein phosphorylation (GO:0006468), protein autophosphorylation (GO:0046777): ACCEPT/KEEP — autophosphorylation is a real PTM but mechanistic detail; protein phosphorylation (of RBOHD/OSCA1.3/FLS2/BAK1/WRKY/ER) is core.
PTI/immune signaling BP terms (GO:0002221, GO:0002237, GO:0042742, GO:0050832, GO:1900424): ACCEPT (core immune function).
Stomatal movement (GO:0010119): ACCEPT/KEEP (real, OSCA1.3-RBOHD pathway).
Hormone regulation (GO:0080141 JA, GO:0080142 SA), response to fungus (GO:0009620): KEEP_AS_NON_CORE (downstream/regulatory, supported but not the primary biochemical function).
PM (GO:0005886), endosome/endomembrane (GO:0010008, GO:0005768, GO:0012505), nucleus (GO:0005634): ACCEPT/KEEP (supported by focused studies).
Nucleolus (GO:0005730): MARK_AS_OVER_ANNOTATED — only from a generic GFP-ORF overexpression screen, never substantiated; biologically implausible for a membrane-anchored kinase.
Cytoplasm (GO:0005737), Golgi (GO:0005794): KEEP_AS_NON_CORE / MARK_AS_OVER_ANNOTATED — generic high-throughput, weakly supported.
Mitochondrion (GO:0005739, ISM): REMOVE — pure sequence-model prediction (AtSubP), contradicted by PM lipid-anchor and all experimental localization data.
protein binding (GO:0005515, IPI x many): KEEP_AS_NON_CORE / MARK_AS_OVER_ANNOTATED — real interactions but uninformative as bare MF; specifics captured elsewhere.

📄 View Raw YAML

id: O48814
gene_symbol: BIK1
product_type: PROTEIN
status: INITIALIZED
taxon:
  id: NCBITaxon:3702
  label: Arabidopsis thaliana
description: BIK1 (BOTRYTIS-INDUCED KINASE 1) is a plasma membrane-anchored receptor-like cytoplasmic kinase (RLCK subfamily VII) of Arabidopsis thaliana and a catalytically active dual-specificity Ser/Thr protein kinase (EC 2.7.11.1). It is tethered to the inner leaflet of the plasma membrane via N-terminal myristoylation (Gly-2) and palmitoylation (Cys-4), where it constitutively associates with pattern-recognition receptor (PRR) complexes such as FLS2-BAK1, EFR-BAK1, PEPR1 and MIK2. Upon perception of pathogen-/damage-associated molecular patterns (PAMPs/DAMPs, e.g. flg22, elf18, AtPep1, SCOOP peptides), BIK1 is rapidly trans-phosphorylated in a BAK1- and PRR-dependent manner, dissociates from the receptor complex, and relays immune signaling by directly phosphorylating downstream substrates. Key substrates include the NADPH oxidase RBOHD (driving the apoplastic reactive oxygen species burst in a calcium-independent manner) and the calcium-permeable channel OSCA1.3 (promoting cytosolic Ca2+ influx and stomatal closure). BIK1 thereby functions as a central convergence node of PAMP-triggered immunity (PTI), controlling the ROS burst, calcium signaling, stomatal immunity, and defense against bacterial and fungal pathogens. A pool of activated, phosphorylated BIK1 relocates to the nucleus where it phosphorylates WRKY transcription factors to regulate jasmonic acid and salicylic acid defense-hormone homeostasis, and a portion is internalized into endosomes following ligand-induced monoubiquitination. BIK1 activity is tightly tuned by the phosphatase PP2C38, the calcium-dependent kinase CPK28 (turnover), and the MAP4K SIK1 (stabilization), and is inactivated by the Xanthomonas effector AvrAC/XopAC via uridylylation of activation-loop residues. Beyond immunity, BIK1 negatively regulates brassinosteroid signaling (via BRI1), modulates ethylene signaling (via EIN3), and contributes to growth and developmental processes (root hair growth, leaf and inflorescence architecture via ERECTA), reflecting its role in the growth-defense tradeoff.
existing_annotations:
- term:
    id: GO:0004672
    label: protein kinase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: BIK1 is a catalytically active protein kinase; this InterPro-based electronic annotation is correct but less specific than the experimentally supported Ser/Thr kinase terms.
    action: MODIFY
    reason: Correct but general. BIK1 is a proven Ser/Thr (dual-specificity) protein kinase (EC 2.7.11.1); the more specific term protein serine/threonine kinase activity is preferred and is independently supported by experimental annotations.
    proposed_replacement_terms:
    - id: GO:0004674
      label: protein serine/threonine kinase activity
    supported_by:
    - reference_id: PMID:24104392
      supporting_text: "we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time."
- term:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000003
  qualifier: enables
  review:
    summary: Electronic EC-based annotation of Ser/Thr protein kinase activity; correct and a core molecular function of BIK1.
    action: ACCEPT
    reason: BIK1 is an experimentally validated Ser/Thr protein kinase (EC 2.7.11.1) that phosphorylates RBOHD, OSCA1.3, FLS2, BAK1 and WRKY transcription factors. The electronic EC mapping correctly captures the core catalytic function.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "This phosphorylation is dependent on BIK1 kinase activity, since a kinase-dead variant, GST–BIK1(KD), did not phosphorylate OSCA1.3-loop1"
- term:
    id: GO:0005524
    label: ATP binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000002
  qualifier: enables
  review:
    summary: ATP binding is required for BIK1 kinase catalysis; supported by a defined ATP-binding site and an ATP-binding Lys-105 whose mutation abolishes activity.
    action: ACCEPT
    reason: BIK1 has a canonical protein kinase ATP-binding pocket (Gly-rich loop 73-81, catalytic Lys-105). Mutation of Lys-105 (with Lys-106) gives a kinase-dead enzyme, confirming functional ATP binding. Standard supporting MF for an active kinase.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "ATP-binding Lys-105; active-site (proton acceptor) Asp-202."
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: Nuclear localization is supported by a focused study showing activation-dependent relocation of BIK1 to the nucleus; this electronic subcellular-location annotation is corroborated experimentally.
    action: ACCEPT
    reason: A pool of phosphorylated/activated BIK1 moves to the nucleus and phosphorylates WRKY transcription factors. Although BIK1 is predominantly plasma membrane-anchored, nuclear localization is a genuine, mechanistically defined secondary location.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation."
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: Plasma membrane is the principal location of BIK1; this electronic annotation is strongly supported by lipid-anchoring and multiple focused studies.
    action: ACCEPT
    reason: BIK1 is myristoylated (Gly-2) and palmitoylated (Cys-4) and resides at the plasma membrane where it associates with PRR complexes. Core location for its immune signaling function.
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response."
- term:
    id: GO:0010008
    label: endosome membrane
  evidence_type: IEA
  original_reference_id: GO_REF:0000044
  qualifier: located_in
  review:
    summary: BIK1 is internalized into endocytic compartments after ligand-induced monoubiquitination; endosome membrane localization is experimentally supported.
    action: KEEP_AS_NON_CORE
    reason: Endosomal localization reflects ligand-induced internalization of BIK1 with FLS2, a regulated trafficking step downstream of activation rather than the primary site of catalytic immune signaling.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "supported by ligand-induced internalization study"
- term:
    id: GO:0106310
    label: protein serine kinase activity
  evidence_type: IEA
  original_reference_id: GO_REF:0000116
  qualifier: enables
  review:
    summary: Rhea/EC-based electronic annotation of protein serine kinase activity; consistent with experimentally proven BIK1 catalytic activity.
    action: ACCEPT
    reason: BIK1 phosphorylates serine residues on substrates such as RBOHD (S39/S339/ S343) and OSCA1.3 (S54). The electronic annotation correctly captures part of the core kinase activity; it is independently supported by EXP annotations.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "BIK1 predominantly phosphorylates S54"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20018686
  qualifier: enables
  review:
    summary: Bare protein binding from the BIK1-FLS2/BAK1 interaction study; uninformative as a molecular function although the interaction itself is real and central.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Per curation guidelines, bare GO:0005515 protein binding is uninformative. The underlying interaction (BIK1 associating with and trans-phosphorylating the FLS2/BAK1 receptor complex) is genuine and is captured by the kinase activity MF and the PRR signaling BP terms; the bare binding term should not be treated as a core function."
    supported_by:
    - reference_id: PMID:20018686
      supporting_text: "BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro."
- term:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  evidence_type: TAS
  original_reference_id: PMID:30584105
  qualifier: enables
  review:
    summary: Traceable assertion of Ser/Thr protein kinase activity; correct and a core molecular function of BIK1.
    action: ACCEPT
    reason: BIK1 is an experimentally validated Ser/Thr protein kinase. This is one of several annotations capturing the core catalytic function and is well supported.
    supported_by:
    - reference_id: PMID:24104392
      supporting_text: "Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity."
    - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
      supporting_text: "Experimental data support BIK1 as an **active serine/threonine protein kinase**"
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: EXP
  original_reference_id: PMID:26021844
  qualifier: located_in
  review:
    summary: Experimental support for plasma membrane localization via myristoylation at Gly-2; core location.
    action: ACCEPT
    reason: This study mapped N-myristoylation at Gly-2 and showed that Gly-2 mutation drastically reduces plasma membrane localization, directly demonstrating lipid-anchored PM residence. Core to BIK1 function.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "Drastic reduction of plasma membrane localization and strong increase of cytoplasmic localization."
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: EXP
  original_reference_id: PMID:32404997
  qualifier: located_in
  review:
    summary: Experimental support for plasma membrane localization where BIK1 resides with FLS2 before ligand-induced internalization; core location.
    action: ACCEPT
    reason: BIK1 is monoubiquitinated and internalized from the plasma membrane upon flg22 perception, establishing the PM as the resting site of BIK1 within the receptor complex. Core location.
    supported_by:
    - reference_id: PMID:32404997
      supporting_text: "Ligand-induced monoubiquitination of BIK1 regulates plant immunity."
- term:
    id: GO:0010008
    label: endosome membrane
  evidence_type: EXP
  original_reference_id: PMID:32404997
  qualifier: located_in
  review:
    summary: Experimentally supported endosome membrane localization following ligand-induced internalization; a regulated downstream trafficking step.
    action: KEEP_AS_NON_CORE
    reason: BIK1 internalizes into endocytic compartments after monoubiquitination upon flg22 perception. Real but a downstream regulatory localization, not the primary site of signaling activity.
    supported_by:
    - reference_id: PMID:32404997
      supporting_text: "Ligand-induced monoubiquitination of BIK1 regulates plant immunity."
- term:
    id: GO:0106310
    label: protein serine kinase activity
  evidence_type: EXP
  original_reference_id: PMID:24104392
  qualifier: enables
  review:
    summary: Experimentally demonstrated protein serine kinase activity (and dual-specificity autophosphorylation); core molecular function.
    action: ACCEPT
    reason: This study profiled BIK1 autophosphorylation, demonstrating serine phosphorylation activity and establishing BIK1 as a dual-specificity kinase. Core catalytic function.
    supported_by:
    - reference_id: PMID:24104392
      supporting_text: "we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time."
- term:
    id: GO:0106310
    label: protein serine kinase activity
  evidence_type: EXP
  original_reference_id: PMID:32846426
  qualifier: enables
  review:
    summary: Experimentally demonstrated serine kinase activity toward OSCA1.3 (S54); core molecular function.
    action: ACCEPT
    reason: BIK1 directly phosphorylates the OSCA1.3 channel at Ser-54 in a kinase-activity-dependent manner, directly demonstrating protein serine kinase activity on a physiological substrate. Core catalytic function.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "BIK1 predominantly phosphorylates S54"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:31803215
  qualifier: enables
  review:
    summary: Bare protein binding from the BIK1-ERECTA developmental study; uninformative MF although the interaction (and phosphorylation of ER) is real.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The specific relationship (BIK1 interacts with and phosphorylates ERECTA-family RLKs in leaf/inflorescence development) is a genuine kinase-substrate interaction captured by the kinase MF; the bare binding term is not a core function."
    supported_by:
    - reference_id: PMID:31803215
      supporting_text: "BIK1 interacts with ER-family proteins and directly phosphorylates ER."
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: HDA
  original_reference_id: PMID:15610358
  qualifier: located_in
  review:
    summary: Nucleus from a generic high-throughput GFP-ORF screen; the location is independently corroborated by a focused activation-dependent study so it is accepted.
    action: ACCEPT
    reason: Although this HDA call comes from a generic GFP-ORF overexpression localization screen rather than a focused BIK1 study, nuclear localization is independently and directly demonstrated (PMID:29649442) as a regulated, activation-dependent location where BIK1 phosphorylates WRKY TFs.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors"
- term:
    id: GO:0005730
    label: nucleolus
  evidence_type: HDA
  original_reference_id: PMID:15610358
  qualifier: located_in
  review:
    summary: Nucleolar localization derives only from a generic GFP-ORF overexpression screen and is not substantiated by any focused BIK1 study; likely an over-annotation.
    action: MARK_AS_OVER_ANNOTATED
    reason: "The nucleolus call comes from a high-throughput GFP-ORF transient overexpression screen that classified proteins into broad categories including nucleolar. It is biologically implausible for a myristoylated/palmitoylated, membrane-anchored immune kinase and is not corroborated by any dedicated BIK1 study (focused work supports plasma membrane, nucleus and endosomes only)."
    supported_by:
    - reference_id: PMID:15610358
      supporting_text: "These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments."
- term:
    id: GO:0005737
    label: cytoplasm
  evidence_type: HDA
  original_reference_id: PMID:15610358
  qualifier: located_in
  review:
    summary: Cytoplasm from a generic GFP-ORF overexpression screen; weakly supported and likely an artifact of overexpression, kept as non-core.
    action: KEEP_AS_NON_CORE
    reason: The cytoplasmic HDA call is from a generic overexpression localization screen. A cytoplasmic pool can appear when membrane targeting (myristoylation/ palmitoylation) is overwhelmed or disrupted (e.g. Gly-2 mutation increases cytoplasmic signal), so it is biologically plausible but not a core functional location; retained as non-core.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "strong increase of cytoplasmic localization"
- term:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  evidence_type: IDA
  original_reference_id: PMID:32846426
  qualifier: enables
  review:
    summary: Direct experimental demonstration of Ser/Thr protein kinase activity (phosphorylation of OSCA1.3); core molecular function.
    action: ACCEPT
    reason: In vitro radioactive kinase assays show BIK1 directly phosphorylates OSCA1.3 in a kinase-activity-dependent manner, directly demonstrating the core Ser/Thr kinase function.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "This phosphorylation is dependent on BIK1 kinase activity, since a kinase-dead variant, GST–BIK1(KD), did not phosphorylate OSCA1.3-loop1"
- term:
    id: GO:0006468
    label: protein phosphorylation
  evidence_type: IDA
  original_reference_id: PMID:32846426
  qualifier: acts_upstream_of_or_within
  review:
    summary: Protein phosphorylation as the biological process by which BIK1 transmits immune signals (e.g. phosphorylating OSCA1.3); core activity.
    action: ACCEPT
    reason: BIK1 transduces immune signals by phosphorylating downstream substrates. The BP term protein phosphorylation correctly captures this; directly supported by OSCA1.3 phosphorylation.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22"
- term:
    id: GO:0010119
    label: regulation of stomatal movement
  evidence_type: IGI
  original_reference_id: PMID:32846426
  qualifier: acts_upstream_of_or_within
  review:
    summary: BIK1 controls stomatal closure during immunity via phosphorylation of the OSCA1.3 Ca2+ channel; well-supported regulatory role.
    action: ACCEPT
    reason: BIK1-mediated phosphorylation of OSCA1.3 is required for flg22-induced stomatal closure (stomatal immunity), and BIK1/RBOHD also controls stomatal movement. A genuine, mechanistically defined role in regulating stomatal movement in the immune context.
    supported_by:
    - reference_id: PMID:32846426
      supporting_text: "OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling"
    - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
      supporting_text: "BIK1 (with PBL1) is required for MAMP/DAMP-induced calcium elevations, positioning BIK1 in early signaling outputs beyond ROS."
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: enables
  review:
    summary: Bare protein binding from the monoubiquitination study (BIK1 binds FLS2 and the ATL44/ATL45 ubiquitin ligases); uninformative MF.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The underlying interactions (BIK1 with FLS2 and with the RING E3 ligases ATL44/RHA3A and ATL45/RHA3B) are real and are captured by the PRR signaling and ubiquitination-related biology described in the notes; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "monoubiquitination of BIK1 by ATL44/RHA3A and ATL45/RHA3B"
- term:
    id: GO:0012505
    label: endomembrane system
  evidence_type: IDA
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: located_in
  review:
    summary: Endomembrane system localization reflecting ligand-induced internalization of BIK1; correct but more specific endosome terms are also annotated.
    action: KEEP_AS_NON_CORE
    reason: Consistent with BIK1 internalization into endocytic compartments after monoubiquitination. The more specific endosome/endosome membrane terms are also present; this general term is retained as a non-core, downstream trafficking location.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "supported by ligand-induced internalization study"
- term:
    id: GO:0002221
    label: pattern recognition receptor signaling pathway
  evidence_type: IMP
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: involved_in
  review:
    summary: BIK1 is a central component of PRR (FLS2/EFR-BAK1) signaling; core biological process.
    action: ACCEPT
    reason: "BIK1 is the canonical RLCK relaying signaling from cell-surface PRR complexes downstream of PAMP perception. PRR signaling pathway is a core BP for BIK1, supported by loss-of-function phenotypes in immune signaling."
    supported_by:
    - reference_id: PMID:20018686
      supporting_text: "BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling."
    - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
      supporting_text: "BIK1 is repeatedly described as a PRR-associated kinase acting downstream of multiple PRRs (FLS2, EFR; also CERK1, PEPR1), linking PAMP perception to downstream defense signaling."
- term:
    id: GO:0002237
    label: response to molecule of bacterial origin
  evidence_type: IDA
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: involved_in
  review:
    summary: BIK1 mediates responses to bacterial PAMPs such as flagellin (flg22); core immune process.
    action: ACCEPT
    reason: BIK1 is phosphorylated and monoubiquitinated specifically in response to the bacterial PAMP flg22 and is required for the downstream immune response. The term (which explicitly covers flagellin-derived peptides) is a core BP for BIK1.
    supported_by:
    - reference_id: PMID:20018686
      supporting_text: "BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1."
- term:
    id: GO:0005768
    label: endosome
  evidence_type: IDA
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: located_in
  review:
    summary: Endosome localization from ligand-induced internalization of BIK1; experimentally supported downstream trafficking location.
    action: KEEP_AS_NON_CORE
    reason: BIK1 internalizes into endosomes after monoubiquitination upon flg22 perception. A genuine but downstream regulatory localization, not the primary site of signaling activity.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "internalization \"into endocytic compartments\""
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: IDA
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: located_in
  review:
    summary: Plasma membrane localization of BIK1 within the FLS2 complex prior to internalization; core location.
    action: ACCEPT
    reason: BIK1 resides at the plasma membrane in the resting receptor complex and is internalized upon ligand perception, confirming the PM as its primary functional location. Core.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "Plasma membrane is the principal, well-supported location"
- term:
    id: GO:0042742
    label: defense response to bacterium
  evidence_type: IMP
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: involved_in
  review:
    summary: BIK1 is required for defense against bacterial pathogens (e.g. Pseudomonas syringae); core immune function.
    action: ACCEPT
    reason: BIK1 ubiquitination mutants show enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000, and BIK1 drives the ROS/Ca2+ outputs that restrict bacterial entry. Defense response to bacterium is a core BP.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "negative regulator of basal resistance to Pst"
    - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
      supporting_text: "BIK1-dependent signaling through RBOHD is linked to stomatal movement that restricts bacterial entry, with impairment when BIK1 function (or its RBOHD phosphorylation) is compromised."
- term:
    id: GO:0050832
    label: defense response to fungus
  evidence_type: IMP
  original_reference_id: DOI:10.1038/s41586-020-2210-3
  qualifier: involved_in
  review:
    summary: BIK1 contributes to defense against fungal pathogens (e.g. Botrytis cinerea); supported immune function.
    action: ACCEPT
    reason: BIK1 was originally identified as required for resistance to necrotrophic fungi, and ubiquitination mutants show enhanced susceptibility to Botrytis cinerea. Defense response to fungus is a genuine, core-adjacent immune BP.
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens"
- term:
    id: GO:0005634
    label: nucleus
  evidence_type: IDA
  original_reference_id: PMID:29649442
  qualifier: located_in
  review:
    summary: Direct experimental evidence that activated BIK1 localizes to the nucleus to phosphorylate WRKY TFs; mechanistically defined secondary location.
    action: ACCEPT
    reason: This focused study directly demonstrated nuclear localization of BIK1 and its interaction with nuclear WRKY transcription factors regulating JA/SA. A genuine activation-dependent location supporting the PRR-BIK1-WRKY axis.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors involved in the JA and salicylic acid (SA) regulation."
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: IDA
  original_reference_id: PMID:29649442
  qualifier: located_in
  review:
    summary: Direct experimental confirmation of plasma membrane localization (the documented resting location of BIK1); core location.
    action: ACCEPT
    reason: This study confirms BIK1's documented plasma membrane localization alongside its activation-dependent nuclear pool. PM is the core functional location.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "in addition to its documented plasma membrane localization, BIK1 also localizes to the nucleus"
- term:
    id: GO:0080141
    label: regulation of jasmonic acid biosynthetic process
  evidence_type: IEP
  original_reference_id: PMID:29649442
  qualifier: involved_in
  review:
    summary: BIK1 regulates JA levels via phosphorylation of WRKY TFs; a downstream regulatory output rather than the core biochemical function.
    action: KEEP_AS_NON_CORE
    reason: BIK1 modulates JA homeostasis through the PRR-BIK1-WRKY axis. This is a genuine but downstream/regulatory consequence of BIK1 immune signaling, not its core kinase function; retained as non-core.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1."
- term:
    id: GO:0080141
    label: regulation of jasmonic acid biosynthetic process
  evidence_type: IMP
  original_reference_id: PMID:29649442
  qualifier: involved_in
  review:
    summary: BIK1 phosphosite mutants alter JA levels, supporting a regulatory role in JA biosynthesis; downstream output, non-core.
    action: KEEP_AS_NON_CORE
    reason: Phosphomimetic mutations at EFR-targeted BIK1 sites elevate JA, genetically linking BIK1 to JA regulation. Genuine but a downstream regulatory output of BIK1 signaling, not its core function.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections."
- term:
    id: GO:0080142
    label: regulation of salicylic acid biosynthetic process
  evidence_type: IEP
  original_reference_id: PMID:29649442
  qualifier: involved_in
  review:
    summary: BIK1 regulates SA homeostasis via the PRR-BIK1-WRKY axis; downstream regulatory output, non-core.
    action: KEEP_AS_NON_CORE
    reason: BIK1 modulates SA levels through phosphorylation of WRKY transcription factors. A genuine downstream regulatory consequence of BIK1 signaling, retained as non-core. (BIK1 was also originally shown to influence SA accumulation.)
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "WRKY transcription factors involved in the JA and salicylic acid (SA) regulation"
- term:
    id: GO:0080142
    label: regulation of salicylic acid biosynthetic process
  evidence_type: IMP
  original_reference_id: PMID:29649442
  qualifier: involved_in
  review:
    summary: BIK1 phosphosite mutants alter SA-associated defense outputs; downstream regulatory role, non-core.
    action: KEEP_AS_NON_CORE
    reason: Mutations at EFR-targeted BIK1 phosphosites change hormone levels and bacterial resistance, genetically linking BIK1 to SA regulation. Downstream output of BIK1 signaling, retained as non-core.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "Phosphomimetic mutations of these sites resulted in increased phytohormones and enhanced resistance to bacterial infections."
- term:
    id: GO:0002237
    label: response to molecule of bacterial origin
  evidence_type: IMP
  original_reference_id: PMID:25522736
  qualifier: involved_in
  review:
    summary: BIK1 is required for calcium responses to bacterial PAMPs (flg22, elf18); core immune process.
    action: ACCEPT
    reason: BIK1 (with PBL1) is genetically required for MAMP/DAMP-induced calcium elevations triggered by bacteria-derived flg22 and elf18. Core response to bacterial molecules.
    supported_by:
    - reference_id: PMID:25522736
      supporting_text: "is also required for MAMP/DAMP-induced calcium elevations."
- term:
    id: GO:0002237
    label: response to molecule of bacterial origin
  evidence_type: IMP
  original_reference_id: PMID:29649442
  qualifier: involved_in
  review:
    summary: BIK1 mediates responses to bacterial PAMPs downstream of EFR/FLS2; core immune process.
    action: ACCEPT
    reason: BIK1 is phosphorylated by the PRR EFR upon perception of bacterial elf18 and is required for elf18/flg22-triggered responses. Core response to bacterial molecules.
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "EFR regulates the phytohormone jasmonic acid (JA) through direct phosphorylation of a receptor-like cytoplasmic kinase, BIK1."
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:29649442
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-EFR and BIK1-WRKY interaction study; uninformative MF although the interactions are biologically central.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The underlying interactions (BIK1 with the PRR EFR and with nuclear WRKY transcription factors WRKY33/50/51/57) are real and are captured by the kinase MF and the nuclear hormone-regulation BP terms; bare binding is not a core function."
    supported_by:
    - reference_id: PMID:29649442
      supporting_text: "BIK1 also localizes to the nucleus and interacts directly with WRKY transcription factors"
- term:
    id: GO:1900424
    label: regulation of defense response to bacterium
  evidence_type: IMP
  original_reference_id: PMID:25522736
  qualifier: involved_in
  review:
    summary: BIK1 regulates antibacterial defense via control of early MAMP-induced calcium signaling; supported immune function.
    action: ACCEPT
    reason: BIK1 is required for MAMP/DAMP-induced calcium signaling that initiates antibacterial defense responses, placing it as a regulator of defense response to bacterium. Core-adjacent immune BP.
    supported_by:
    - reference_id: PMID:25522736
      supporting_text: "is also required for MAMP/DAMP-induced calcium elevations."
- term:
    id: GO:0005794
    label: Golgi apparatus
  evidence_type: HDA
  original_reference_id: PMID:28887381
  qualifier: located_in
  review:
    summary: Golgi localization derives only from a global membrane-protein correlation-profiling proteomics survey, not a focused BIK1 study; likely over-annotation.
    action: MARK_AS_OVER_ANNOTATED
    reason: "This HDA call is from a high-throughput protein correlation profiling proteomics study of membrane-protein oligomerization, not a dedicated BIK1 localization study. Golgi residence is not supported by any focused BIK1 work (which establishes plasma membrane, nucleus and endosomes), and likely reflects fractionation/co-migration rather than genuine steady-state Golgi localization."
    supported_by:
    - reference_id: PMID:28887381
      supporting_text: "Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling."
- term:
    id: GO:0002221
    label: pattern recognition receptor signaling pathway
  evidence_type: IMP
  original_reference_id: PMID:21862710
  qualifier: acts_upstream_of_or_within
  review:
    summary: BIK1 is genetically and biochemically required for PAMP-triggered immunity downstream of PRRs; core biological process.
    action: ACCEPT
    reason: This study established biochemical/genetic requirements for BIK1 in PAMP-triggered immunity (and growth/ethylene signaling). PRR signaling pathway is a core BP for BIK1.
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "Central RLCK that relays PTI signaling from PRR–BAK1 complexes"
- term:
    id: GO:0005739
    label: mitochondrion
  evidence_type: ISM
  original_reference_id: GO_REF:0000122
  qualifier: located_in
  review:
    summary: Mitochondrial localization is a pure sequence-model prediction (AtSubP) with no experimental support and is contradicted by BIK1 biology.
    action: REMOVE
    reason: "This is an ISM (sequence-model) prediction from AtSubP (GO_REF:0000122) with no experimental backing. BIK1 is N-myristoylated/palmitoylated and anchored to the plasma membrane, with experimentally established nucleus and endosome pools; mitochondrial localization is biologically implausible and contradicted by all focused studies. Demonstrably wrong electronic prediction."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "with no experimental support and is inconsistent with the myristoyl/palmitoyl PM anchor"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20404519
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-FLS2/BAK1 interaction and phosphorylation study; uninformative MF although the interaction is central.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The interaction (BIK1 with FLS2 and BAK1, with mutual trans-phosphorylation) is genuine and captured by the kinase MF and PRR signaling BP; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "FLS2/BAK1 (PMID:20018686, PMID:20404519, PMID:20413097)"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:20413097
  qualifier: enables
  review:
    summary: Bare protein binding from study showing BIK1 integrates signaling from multiple immune receptors and is an effector target; uninformative MF.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The biology (BIK1 associating with multiple PRRs and being targeted by a Pseudomonas effector) is captured by the PRR signaling and defense BP terms; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "BIK1 integrates signaling from multiple immune receptors and is targeted by a Pseudomonas effector"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:23431184
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-PEPR1 interaction study (ethylene-induced immunity); uninformative MF although the interaction is real.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The specific interaction (BIK1 with the PEPR1 receptor, mediating Pep1/ethylene-induced immunity) is genuine but is better captured by signaling BP terms; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "PEPR1 (PMID:23431184)"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:23818580
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-BRI1 interaction study (brassinosteroid signaling); uninformative MF although the interaction is real.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The specific interaction (BIK1 with the BR receptor BRI1, negatively regulating BR signaling) is genuine but better captured by signaling BP terms; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "Negative regulator of brassinosteroid signaling via BRI1 interaction"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:24629339
  qualifier: enables
  review:
    summary: Bare protein binding from the BIK1-RBOHD study; uninformative as bare MF, but the kinase-substrate relationship is a core part of BIK1 function.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The underlying interaction (BIK1 binding and directly phosphorylating the NADPH oxidase RBOHD to drive the ROS burst) is a core function captured by the kinase MF and ROS/defense BP terms; bare binding should not stand as a separate functional claim."
    supported_by:
    - reference_id: PMID:24629339
      supporting_text: "directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation."
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:25525792
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-CPK28 interaction study (regulation of BIK1 turnover); uninformative MF although the interaction is real.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The specific interaction (CPK28 binding BIK1 to buffer immunity and regulate BIK1 turnover) is a genuine regulatory relationship better described by the regulation biology; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "CPK28 (PMID:25525792)"
- term:
    id: GO:0005515
    label: protein binding
  evidence_type: IPI
  original_reference_id: PMID:27494702
  qualifier: enables
  review:
    summary: Bare protein binding from BIK1-PP2C38 interaction study (negative regulation of BIK1); uninformative MF although the interaction is real.
    action: MARK_AS_OVER_ANNOTATED
    reason: "Bare GO:0005515 is uninformative. The specific interaction (the phosphatase PP2C38 binding and negatively regulating BIK1 activation) is a genuine regulatory relationship; bare binding is not a core function."
    supported_by:
    - reference_id: file:ARATH/BIK1/BIK1-notes.md
      supporting_text: "PP2C38 (PMID:27494702)"
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: IDA
  original_reference_id: PMID:16339855
  qualifier: located_in
  review:
    summary: Original demonstration that BIK1 is membrane-localized; core location.
    action: ACCEPT
    reason: The founding BIK1 study showed membrane localization, consistent with its lipid anchoring and role in early pathogen signal transduction. Core location.
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "BIK1 is membrane-localized, suggesting possible involvement in early stages of the recognition or transduction of pathogen response."
- term:
    id: GO:0009620
    label: response to fungus
  evidence_type: IEP
  original_reference_id: PMID:16339855
  qualifier: acts_upstream_of_or_within
  review:
    summary: BIK1 transcript is induced by Botrytis infection; expression-based response to fungus, kept as non-core relative to the more specific defense term.
    action: KEEP_AS_NON_CORE
    reason: This IEP annotation reflects transcriptional induction of BIK1 by Botrytis cinerea. It is supported but is an expression-based (IEP) inference; the more specific defense response to fungus (IMP) better captures the functional role.
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "the Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) gene that is transcriptionally regulated by Botrytis cinerea infection."
- term:
    id: GO:0016301
    label: kinase activity
  evidence_type: IDA
  original_reference_id: PMID:16339855
  qualifier: enables
  review:
    summary: General kinase activity; correct but less specific than the proven protein Ser/Thr kinase activity.
    action: MODIFY
    reason: BIK1 is a protein Ser/Thr (dual-specificity) kinase. The generic kinase activity term should be replaced by the specific protein serine/threonine kinase activity term, which is independently and experimentally supported.
    proposed_replacement_terms:
    - id: GO:0004674
      label: protein serine/threonine kinase activity
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "BIK1 encodes a regulatory protein, specifically a protein kinase, predicted to be specific to Ser/Thr residues"
- term:
    id: GO:0046777
    label: protein autophosphorylation
  evidence_type: IDA
  original_reference_id: PMID:16339855
  qualifier: acts_upstream_of_or_within
  review:
    summary: BIK1 autophosphorylates on numerous Ser/Thr/Tyr residues; a real activity but a mechanistic/PTM detail rather than the primary biological role.
    action: KEEP_AS_NON_CORE
    reason: Autophosphorylation is experimentally well documented (19 in vitro autophosphorylation sites, including phosphotyrosines) and contributes to BIK1 activation, but it is a self-directed catalytic detail; the core function is trans-phosphorylation of downstream immune substrates. Retained as non-core.
    supported_by:
    - reference_id: PMID:24104392
      supporting_text: "we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites"
- term:
    id: GO:0050832
    label: defense response to fungus
  evidence_type: IMP
  original_reference_id: PMID:16339855
  qualifier: acts_upstream_of_or_within
  review:
    summary: bik1 mutants are severely susceptible to necrotrophic fungi, establishing a genuine role in defense response to fungus; core-adjacent immune function.
    action: ACCEPT
    reason: Loss of BIK1 causes severe susceptibility to the necrotrophic fungi Botrytis cinerea and Alternaria brassicicola, directly demonstrating a required role in antifungal defense.
    supported_by:
    - reference_id: PMID:16339855
      supporting_text: "Inactivation of BIK1 causes severe susceptibility to necrotrophic fungal pathogens"
core_functions:
- description: Acts as a dual-specificity (predominantly Ser/Thr) protein kinase that, upon trans-phosphorylation by BAK1 within ligand-activated PRR complexes, directly phosphorylates downstream immune substrates to relay PAMP-triggered immunity signaling.
  supported_by:
  - reference_id: PMID:20018686
    supporting_text: "BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling."
  - reference_id: PMID:24104392
    supporting_text: "we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time."
  - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
    supporting_text: "connecting PRR activation to early defense outputs such as reactive oxygen species"
  molecular_function:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  directly_involved_in:
  - id: GO:0002221
    label: pattern recognition receptor signaling pathway
  - id: GO:0002237
    label: response to molecule of bacterial origin
  locations:
  - id: GO:0005886
    label: plasma membrane
- description: Directly phosphorylates the NADPH oxidase RBOHD in a calcium-independent manner to drive the apoplastic reactive oxygen species (ROS) burst that underlies antibacterial immunity and stomatal defense.
  supported_by:
  - reference_id: PMID:24629339
    supporting_text: "directly phosphorylates the NADPH oxidase RbohD at specific sites in a calcium-independent manner to enhance ROS generation."
  - reference_id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
    supporting_text: "BIK1 directly interacts with and phosphorylates RBOHD following PAMP perception."
  molecular_function:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  directly_involved_in:
  - id: GO:0042742
    label: defense response to bacterium
  locations:
  - id: GO:0005886
    label: plasma membrane
- description: Phosphorylates and activates the calcium-permeable channel OSCA1.3 to promote PAMP-induced cytosolic Ca2+ influx and stomatal closure during immune signaling.
  supported_by:
  - reference_id: PMID:32846426
    supporting_text: "OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling"
  - reference_id: PMID:25522736
    supporting_text: "is also required for MAMP/DAMP-induced calcium elevations."
  molecular_function:
    id: GO:0004674
    label: protein serine/threonine kinase activity
  directly_involved_in:
  - id: GO:0010119
    label: regulation of stomatal movement
  locations:
  - id: GO:0005886
    label: plasma membrane
suggested_questions:
- question: What determines the partitioning of BIK1 between the plasma membrane, nucleus, and endosomes, and how is the activation-dependent nuclear pool sized and timed relative to the ROS/Ca2+ outputs at the membrane?
  experts:
  - Plant immune signaling researchers
  - Cell biologists
- question: How is substrate specificity of BIK1 (RBOHD vs OSCA1.3 vs WRKYs vs receptor kinases) encoded, given its many phosphorylation sites and dual-specificity activity?
  experts:
  - Plant kinase biochemists
  - Structural biologists
- question: To what extent are BIK1's developmental roles (root hair growth, leaf/ inflorescence architecture via ERECTA, BR signaling via BRI1) separable from its immune signaling function in the growth-defense tradeoff?
  experts:
  - Plant developmental biologists
suggested_experiments:
- hypothesis: BIK1 nuclear relocation is required specifically for hormone-branch immunity but dispensable for the membrane ROS/Ca2+ burst.
  description: Engineer nuclear-excluded (membrane-tethered) and constitutively nuclear BIK1 variants and quantify the ROS burst, Ca2+ influx, stomatal closure, JA/SA levels, and bacterial resistance for each, separating membrane from nuclear outputs.
- hypothesis: Distinct BIK1 phosphosites gate distinct downstream substrates.
  description: Systematically combine activation-loop and Tyr phosphosite mutants with in vitro and in vivo phosphorylation assays against RBOHD, OSCA1.3, FLS2/BAK1 and WRKY substrates to map a site-to-substrate code.
references:
- id: file:ARATH/BIK1/BIK1-deep-research-falcon.md
  title: "Falcon/Edison deep research report: BIK1"
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "AI-generated (Falcon/Edison) deep-research synthesis used as supporting context; trace individual claims to primary literature before treating as definitive."
- id: GO_REF:0000002
  title: Gene Ontology annotation through association of InterPro records with GO terms
  findings: []
- id: GO_REF:0000003
  title: Gene Ontology annotation based on Enzyme Commission mapping
  findings: []
- id: GO_REF:0000044
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt
  findings: []
- id: GO_REF:0000116
  title: Automatic Gene Ontology annotation based on Rhea mapping
  findings: []
- id: GO_REF:0000122
  title: AtSubP analysis
  findings: []
- id: DOI:10.1038/s41586-020-2210-3
  title: Ligand-induced monoubiquitination of BIK1 regulates plant immunity.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "DOI resolves to Ma et al. 2020 Nature (= PMID:32404997, also in this reference list). Establishes flg22-induced monoubiquitination of BIK1 by ATL44/RHA3A and ATL45/RHA3B, dissociation from FLS2 complex, internalization into endosomes, and requirement for ROS and antibacterial immunity."
- id: PMID:15610358
  title: High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: "PubMed-verified high-throughput GFP-ORF transient-expression localization screen of 155 fusion proteins (cytoplasmic/nuclear/nucleolar/ organellar/endomembrane categories). Generic overexpression assay that bypasses native N-myristoylation/palmitoylation membrane targeting; source of the cytoplasm/nucleus/nucleolus HDA annotations. The nucleolus call is not substantiated by any focused BIK1 study and is biologically implausible for a membrane-anchored kinase."
- id: PMID:16339855
  title: The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "Full text available and PubMed-verified. Founding BIK1 paper: identifies BIK1 as a membrane-anchored RLCK required for resistance to necrotrophic fungi, negative regulator of basal resistance to Pst, SA modulation, and root hair growth. Source of the membrane, response to fungus, kinase activity, autophosphorylation and defense response to fungus annotations."
- id: PMID:20018686
  title: A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "PubMed-verified (abstract-only in cache). Establishes BIK1 as the RLCK that associates with FLS2/BAK1 and is rapidly phosphorylated upon flagellin perception, then trans-phosphorylates FLS2/BAK1 to propagate immune signaling. Anchors the core PRR-signaling and bacterial-response annotations."
- id: PMID:20404519
  title: Phosphorylation of receptor-like cytoplasmic kinases by bacterial flagellin.
  findings: []
- id: PMID:20413097
  title: Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector.
  findings: []
- id: PMID:21862710
  title: Biochemical and genetic requirements for function of the immune response regulator BOTRYTIS-INDUCED KINASE1 in plant growth, ethylene signaling, and PAMP-triggered immunity in Arabidopsis.
  findings: []
- id: PMID:23431184
  title: BIK1 interacts with PEPRs to mediate ethylene-induced immunity.
  findings: []
- id: PMID:23818580
  title: Inverse modulation of plant immune and brassinosteroid signaling pathways by the receptor-like cytoplasmic kinase BIK1.
  findings: []
- id: PMID:24104392
  title: Identification and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1.
  findings: []
- id: PMID:24629339
  title: The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "PubMed-verified (abstract-only in cache). Establishes the core BIK1->RBOHD->ROS axis: BIK1 directly phosphorylates RBOHD at specific sites in a calcium-independent manner to enhance the ROS burst and control stomatal defense."
- id: PMID:25522736
  title: Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1.
  findings: []
- id: PMID:25525792
  title: The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover.
  findings: []
- id: PMID:26021844
  title: Regulatory role of BOTRYTIS INDUCED KINASE1 in ETHYLENE INSENSITIVE3-dependent gene expression in Arabidopsis.
  findings: []
- id: PMID:27494702
  title: The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.
  findings: []
- id: PMID:28887381
  title: Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling.
  findings: []
  reference_review:
    relevance: LOW
    correctness: VERIFIED
    review_notes: "PubMed-verified high-throughput membrane-protein correlation-profiling proteomics survey, not a focused BIK1 study. Source of the Golgi HDA annotation, which is not corroborated by any dedicated BIK1 localization work; treated as over-annotation."
- id: PMID:29649442
  title: The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "Full text available and PubMed-verified. Crystal structure (PDB 5TOS) plus demonstration that EFR phosphorylates BIK1 and that activated BIK1 relocates to the nucleus to phosphorylate WRKY TFs regulating JA/SA. Supports nucleus localization and the JA/SA regulation annotations via the PRR-BIK1-WRKY axis."
- id: PMID:30584105
  title: Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.
  findings: []
- id: PMID:31803215
  title: BIK1 and ERECTA Play Opposing Roles in Both Leaf and Inflorescence Development in Arabidopsis.
  findings: []
- id: PMID:32404997
  title: Ligand-induced monoubiquitination of BIK1 regulates plant immunity.
  findings: []
- id: PMID:32846426
  title: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity.
  findings: []
  reference_review:
    relevance: HIGH
    correctness: VERIFIED
    review_notes: "Full text available and PubMed-verified. Establishes BIK1->OSCA1.3 Ca2+ channel axis: BIK1 directly phosphorylates OSCA1.3 (S54) in a kinase-activity-dependent manner to promote Ca2+ influx and stomatal closure during immunity. Supports the kinase-activity, protein phosphorylation, and stomatal movement annotations."

BIK1

Gene Description

Existing Annotations Review

Core Functions

Acts as a dual-specificity (predominantly Ser/Thr) protein kinase that, upon trans-phosphorylation by BAK1 within ligand-activated PRR complexes, directly phosphorylates downstream immune substrates to relay PAMP-triggered immunity signaling.

Directly phosphorylates the NADPH oxidase RBOHD in a calcium-independent manner to drive the apoplastic reactive oxygen species (ROS) burst that underlies antibacterial immunity and stomatal defense.

Phosphorylates and activates the calcium-permeable channel OSCA1.3 to promote PAMP-induced cytosolic Ca2+ influx and stomatal closure during immune signaling.

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Gene Research for GO Annotation Review

Target

UniProt Context

Research Objective

Output Requirements

Gene Research Report for GO Annotation Review: BIK1 (Arabidopsis thaliana; UniProt O48814; At2g39660)

1. Executive summary

2. Molecular function

2.1 Key concepts/definitions (current understanding)

2.2 Core biochemical activity and substrate specificity

2.3 Activation and inactivation mechanisms (maturation/processing)

3. Biological process roles

3.1 Best-supported processes in Arabidopsis (direct evidence)

3.2 Recent developments (2023–2024) refining process interpretation

3.3 Current applications and real-world implementations

4. Cellular localization and complexes

4.1 Best-supported subcellular localization

4.2 Complexes and direct interactors

5. Annotation-risk assessment (core vs context-specific; over-extension risks)

5.1 High-confidence “core function” annotations (recommended emphasis)

5.2 Context-specific regulatory annotations (valid but should be framed as regulation of BIK1 in immunity)

5.3 Over-extension risks / terms to avoid without additional direct evidence

6. Key literature (priority to 2023–2024; includes URLs and publication dates)

High-priority recent primary research (2023–2024)

Foundational mechanistic literature

GO-oriented evidence summary table

📚 Additional Documentation

Notes

BIK1 (BOTRYTIS-INDUCED KINASE 1, AT2G39660, UniProt O48814) — research notes

Identity and architecture

Core function: immune signaling hub downstream of cell-surface PRRs

Core function: phosphorylation of RBOHD and ROS regulation

Core function: phosphorylation of Ca2+ channel OSCA1.3 / stomatal immunity

Defense response / hormone regulation

Subcellular localization assessment

Developmental / growth-defense tradeoff (non-core)

Protein-binding (IPI) annotations

Summary of curation decisions

📄 View Raw YAML