The juxtamembrane domains of Arabidopsis CERK1, BAK1, and FLS2 play a conserved role in chitin‐induced signaling

Arabidopsis thaliana CERK1 is an essential receptor‐like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate that the JM domains of LysM‐RLKs, CERK1, and OsCERK1 play a functionally conserved role in the activation of chitin signaling in Arabidopsis. The C‐termini of the JM domains of both CERK1 and OsCERK1 are indispensable for their function. Moreover, after replacing the JM domain of CERK1 with that of the nonhomologous RLK, BAK1 (CJBa) or FLS2 (CJFl), the chimeric CERK1 receptors maintained their ability to activate chitin signaling in Arabidopsis. Interestingly, the heterologous expression of CJBa and CJFl did not induce cell death in Nicotiana benthamiana leaves. These results suggest that the JM domains of CERK1, BAK1, and FLS2 play a conserved role in chitin signaling via a mechanism not related to sequence homology.     

TL1A increased IL-6 production on fibroblast-like synoviocytes by preferentially activating TNF receptor 2 in rheumatoid arthritis

TNF-like protein 1A (TL1A), a member of tumor necrosis factor family, recognized as a ligand of death receptor 3 (DR3) and decoy receptor 3 (DcR3). The interaction of TL1A and DR3 may participate in the pathogenesis of some autoimmune diseases including rheumatoid arthritis (RA). Our previous results showed that high concentrations of TL1A could be found in synovial and serum in RA patients, and it was correlated with disease severity. In addition, TL1A could promote Th17 differentiation induced by TGF-β and IL-6 and increased the production of IL-17A. In the present study, we found that TL1A could promote the expression of IL-6 on fibroblast-like synoviocytes (FLS) of RA patients via NF-κB and JNK signaling pathway. TL1A-stimulated FLS increased the percentage of Th17 of peripheral blood mononuclear cells (PBMC) in RA via the production of IL-6, a critical cytokine involved in the differentiation of Th17. Moreover, the blocking of tumor necrosis factor receptor 2 (TNFR2) decreased TL1A-stimulated IL-6 production by RA FLS. Our results suggest that TL1A was capable of acting on RA FLS to elevate IL-6 expression, which promoted the production of Th17. More importantly, we showed that TL1A could influence RA FLS through binding to TNFR2 rather than DR3 on FLS, which indicated that the treatment of TNF inhibitors not only blocked the TNF but also suppressed the TL1A in RA patients.     

BAK1 is not a target of the Pseudomonas syringae effector AvrPto

Plant cell surface-localized receptor kinases such as FLS2, EFR, and CERK1 play a crucial role in detecting invading pathogenic bacteria. Upon stimulation by bacterium-derived ligands, FLS2 and EFR interact with BAK1, a receptor-like kinase, to activate immune responses. A number of Pseudomonas syringae effector proteins are known to block immune responses mediated by these receptors. Previous reports suggested that both FLS2 and BAK1 could be targeted by the P. syringae effector AvrPto to inhibit plant defenses. Here, we provide new evidence further supporting that FLS2 but not BAK1 is targeted by AvrPto in plants. The AvrPto-FLS2 interaction prevented the phosphorylation of BIK1, a downstream component of the FLS2 pathway.     

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.     

ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response

The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance. acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA). To further explore the role of ACD6 in the defense response, we cloned and characterized the gene. ACD6 encodes a novel protein with putative ankyrin and transmembrane regions. It is a member of one of the largest uncharacterized gene families in higher plants. Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway. Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH). A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P. syringae infection had reduced SA levels and increased susceptibility. Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P. syringae, and BTH-inducible and/or a low level of spontaneous cell death. Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.     

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.     

LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.     

Plant plasma membrane‐resident receptors: Surveillance for infections and coordination for growth and development

As sessile organisms, plants are exposed to pathogen invasions and environmental fluctuations. To overcome the challenges of their surroundings, plants acquire the potential to sense endogenous and exogenous cues, resulting in their adaptability. Hence, plants have evolved a large collection of plasma membrane-resident receptors, including RECEPTOR-LIKE KINASEs(RLKs) and RECEPTOR-LIKE PROTEINs(RLPs) to perceive those signals and regulate plant growth,development, and immunity. The ability of RLKs and RLPs to recognize distinct ligands relies on diverse categories of extracellular domains evolved. Co-regulatory receptors are often required to associate with RLKs and RLPs to facilitate cellular signal transduction. RECEPTOR-LIKE CYTOPLASMIC KINASEs(RLCKs) also associate with the complex, bifurcating the signal to key signaling hubs, such as MITOGEN-ACTIVATED PROTEIN KINASE(MAPK) cascades, to regulate diverse biological processes. Here, we discuss recent knowledge advances in understanding the roles of RLKs and RLPs in plant growth, development, and immunity, and their connection with co-regulatory receptors, leading to activation of diverse intracellular signaling pathways.     

Ceramide kinase: the first decade

It has been some 20 years since the initial discovery of ceramide 1-phosphate (C1P) and nearly a decade since ceramide kinase (CERK) was cloned. Many studies have shown that C1P is important for membrane biology and for the regulation of membrane-bound proteins, and the CERK enzyme has appeared to be tightly regulated in order to control both ceramide levels and production of C1P. Furthermore, C1P made by CERK has emerged as a genuine signalling entity. However, it represents only part of the C1P pool that is available in the cell, therefore suggesting that alternative unknown C1P-producing mechanisms may also play a role. Recent technological developments for measuring complex sphingolipids in biological samples, together with the availability of Cerk-deficient animals as well as potent CERK inhibitors, have now provided new grounds for investigating C1P biology further. Here, we will review the current understanding of CERK and C1P in terms of biochemistry and functional implications, with particular attention to C1P produced by CERK.     

The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens

Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.     

The Arabidopsis Malectin-Like Leucine-Rich Repeat Receptor-Like Kinase IOS1 Associates with the Pattern Recognition Receptors FLS2 and EFR and Is Critical for Priming of Pattern-Triggered Immunity

Plasma membrane-localized pattern recognition receptors such as FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize microbe-associated molecular patterns (MAMPs) to activate the first layer of plant immunity termed pattern-triggered immunity (PTI). A reverse genetics approach with genes responsive to the priming agent β-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants demonstrated defective PTI responses, notably delayed upregulation of PTI marker genes, lower callose deposition, and mitogen-activated protein kinase activities upon bacterial infection or MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to P. syringae and demonstrated a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and FLS2 and EFR. IOS1 also associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in a ligand-independent manner and positively regulated FLS2/BAK1 complex formation upon MAMP treatment. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a regulatory protein of FLS2- and EFR-mediated signaling that primes PTI activation upon bacterial elicitation.     

Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting

The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)–sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b– and ARA6/Rab F1–positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.     

Perception of the novel MAMP eMax from different Xanthomonas species requires the Arabidopsis receptor-like protein ReMAX and the receptor kinase SOBIR

As part of their innate immune system plants carry a number of pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). In a recently published article¹ we described a novel, proteinaceous MAMP termed eMax (enigmatic MAMP of Xanthomonas) that derives from Xanthomonas and gets recognized by the receptor-like protein ReMAX (RECEPTOR OF eMax) of Arabidopsis thaliana. ReMAX has no ortholog in Nicotiana benthamiana and this species does not respond to eMax even when transformed with ReMAX. However, interfamily transfer of eMax perception was successful with a chimeric form of ReMAX where the C-terminal part of the protein was replaced by the corresponding part of the tomato RLP EIX2 (ETHYLENE INDUCING XYLANASE2). In this addendum we describe the difficulties with the purification and identification of the MAMP eMax and we present data demonstrating that functionality of ReMAX, much like that of related RLPs, depends on the presence of the receptor kinase SOBIR (SUPPRESSOR OF BIR1–1).     

Heterotrimeric G Proteins Serve as a Converging Point in Plant Defense Signaling Activated by Multiple Receptor-Like Kinases

In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G PROTEIN α-SUBUNIT1), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) SUPPRESSOR OF BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing SUPPRESSOR OF BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), respectively. By contrast, G PROTEIN α-SUBUNIT1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals.Receptor-like kinases (RLKs) represent one of the largest protein families in plants and play diverse roles in plant development and stress signaling (Morillo and Tax, 2006). In Arabidopsis (Arabidopsis thaliana), there are over 600 RLKs (Shiu and Bleecker, 2001). Most RLKs contain an extracellular domain, a single transmembrane motif, and a cytoplasmic kinase domain. It is believed that the extracellular domains are involved in ligand recognition that subsequently leads to activation of the cytoplasmic kinase domain. Pathogen-associated molecular pattern (PAMP) receptors FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor (EF)-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) all belong to the RLK family. FLS2 and EFR function as receptors for bacterial flagellin and EF-Tu, respectively (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006), while CERK1 is involved in the perception of chitin, a common component of the fungal cell wall (Miya et al., 2007; Wan et al., 2008). CERK1 was also shown to play an important role in defense against bacterial pathogens (Gimenez-Ibanez et al., 2009). Another RLK, Brassinosteroid Insensitive1-associated receptor kinase1 (BAK1), functions as a coreceptor for FLS2 and EFR (Chinchilla et al., 2007; Heese et al., 2007). In addition, a rice (Oryza sativa) RLK, Xa21, functions as the receptor for a peptide derived from AvrXa21 (Song et al., 1995; Lee et al., 2009).Activation of different PAMP receptors often leads to rapid downstream responses, such as oxidative burst, calcium influx, mitogen-activated protein kinase (MAPK) activations, and the up-regulation of defense gene expression (Boller and Felix, 2009). However, our knowledge of how defense responses are regulated downstream of the RLKs remains limited. Genetic analysis of mutants defective in EFR-mediated PAMP responses showed that endoplasmic reticulum (ER) quality control plays an important role in the accumulation of EFR (Li et al., 2009; Lu et al., 2009; Nekrasov et al., 2009; Saijo et al., 2009). ER-resident chaperones are also required for the accumulation of the tobacco (Nicotiana tabacum) INDUCED RECEPTOR-LIKE KINASE (Caplan et al., 2009). BOTRYTIS-INDUCED KINASE1 (BIK1) encodes a cytoplasmic RLK that directly interacts with FLS2 and likely EFR and CERK1 (Lu et al., 2010; Zhang et al., 2010). Knocking out BIK1 leads to modest reductions of PAMP-induced callose deposition, H₂O₂ accumulation, and pathogen resistance. Recently, a group of redundant calcium-dependent protein kinases (CDPKs) were identified as critical regulators of MAPK-independent defense pathways downstream of FLS2 (Boudsocq et al., 2010).In yeast (Saccharomyces cerevisiae) and metazoans, heterotrimeric G proteins, composed of α-, β-, and γ-subunits, serve as essential signaling intermediates between cell surface G-protein-coupled receptors (GPCRs) and their downstream targets (Temple and Jones, 2007). Binding of ligands to GPCRs leads to the exchange of GDP for GTP in the α-subunit, resulting in the activation of the G protein. In Arabidopsis, there is one Gα subunit (G PROTEIN α-SUBUNIT1 [GPA1]), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3; Temple and Jones, 2007; Chakravorty et al., 2011). Whereas AGG1 and AGG2 are closely related, AGG3 only shares very limited homology to AGG1 and AGG2. GPCR-like proteins have been identified in plants, and their interactions with Gα have been experimentally proven, although their status as bona fide GPCRs remains controversial (Liu et al., 2007; Gookin et al., 2008; Pandey et al., 2009).Heterotrimeric G proteins have been shown to be involved in a wide range of biological processes, including plant immunity (Perfus-Barbeoch et al., 2004). Analysis of rice dwarf1 mutants with defects in the Gα subunit indicates that the rice Gα subunit plays an important role in resistance against rice blast (Suharsono et al., 2002). In Arabidopsis, loss of function of the Gβ or Gγ subunits leads to compromised resistance against the necrotrophic pathogen Plectosphaerella cucumerina (Llorente et al., 2005; Delgado-Cerezo et al., 2012). By contrast, loss of function of the Gα subunit GPA1 results in enhanced resistance against the pathogen (Llorente et al., 2005). Resistance against other necrotrophic pathogens, such as Fusarium oxysporum, Alternaria brassicicola, and Botrytis cinerea, was also found to be compromised in AGB1- and AGG1/AGG2-deficient mutants (Trusov et al., 2006, 2007, 2009). In addition, loss of function of the Gβ subunit leads to reduced elf18-induced resistance against Agrobacterium tumefaciens as well as reduced reactive oxygen species (ROS) production triggered by flagellin22 (flg22) and elf18 (an 18-amino acid peptide that represents the N terminus of bacterial EF-Tu; Ishikawa, 2009). Multiple surface residues of AGB1 were recently shown to play important roles in resistance against necrotrophic pathogens and flg22-induced ROS production (Jiang et al., 2012).Arabidopsis GPA1 was also found to play an important role in stomatal defense. In gpa1 mutants, flg22-induced inhibition of stomatal opening and inward K⁺ channels in guard cells are blocked (Zhang et al., 2008b). Growth of the coronatine-deficient Pseudomonas syringae pv tomato DC3118 is dramatically increased in gpa1 mutant plants (Zeng and He, 2010). In Nicotiana benthamiana, silencing of the Gα and Gβ subunits also leads to reduced elicitor-induced stomatal closure as well as hypersensitive responses induced by harpin (Zhang et al., 2012a).Arabidopsis BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1) encodes a BAK1-associated RLK (Gao et al., 2009). Knocking out BIR1 results in constitutive activation of cell death and defense responses in a manner that is partially dependent on PHYTOALEXIN DEFICIENT4 (PAD4), a positive regulator of resistance mediated by the Toll-Interleukin-1 Receptor-like-Nucleotide-Binding-Leu-Rich Repeat domain class of resistance proteins. To identify signaling components downstream of BIR1, a suppressor screen was performed in the bir1-1 pad4-1 double-mutant background. A number of suppressor of bir1-1 (sobir) mutants suppressing the seedling lethality phenotype of bir1-1 were identified. SOBIR1 encodes another RLK whose overexpression is sufficient to activate cell death and defense responses (Gao et al., 2009). Combining the sobir1-1 and pad4-1 mutations results in complete suppression of cell death and enhanced pathogen resistance in bir1-1, suggesting that SOBIR1 and PAD4 function in parallel to regulate cell death and defense responses. Here, we report the discovery and characterization of SOBIR2, which encodes the Arabidopsis heterotrimeric G-protein β-subunit AGB1 that functions downstream of SOBIR1 to regulate cell death and defense responses.     

The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 is involved in the perception of insect feeding

The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize.     

Adipocyte Lipolysis-stimulated Interleukin-6 Production Requires Sphingosine Kinase 1 Activity

Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of β₃-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes.     

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.     

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.