Extracellular Leucine-rich Repeat Domain Research Articles

Mechanisms of thyrotropin receptor-mediated phenotype variability deciphered by gene mutations and M453T-knockin model.

The clinical spectrum of thyrotropin receptor-mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T-knockin model revealed that the phenotype was dependent on the mutation's signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

HSP40 mediated TLR-Dorsal-AMPs pathway in Portunus trituberculatus

Heat shock protein 40 (HSP40) is a kind of molecular chaperone involved in various immune responses. However, the exact roles of HSP40 in immune defense against bacteria remain largely unclear. In this study, the activation function of a type Ⅰ HSP40 from Portunus trituberculatus (PtHSP40-Ⅰ) in the TLR pathway was investigated. The results showed that PtHSP40-Ⅰ can bind to lipopolysaccharide (LPS) and peptidoglycan (PGN). The PtHSP40-Ⅰ also exhibited binding activity toward the extracellular leucine-rich repeat (LRR) domain of Toll-like receptor (TLR). Moreover, the PtHSP40-Ⅰ could promote the transcription factor Dorsal translocated from cytoplasm into the nucleus in hemocytes and participated in regulating the expression of anti-lipopolysaccharide factor (ALF) and crustin. These findings provided insights into the activation mechanisms of TLR pathway mediated by HSP40 and offered basic theory of disease control in P. trituberculatus aquaculture.

Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Silencing a Simple Extracellular Leucine-Rich Repeat Gene OsI-BAK1 Enhances the Resistance of Rice to Brown Planthopper Nilaparvata lugens.

Many plant proteins with extracellular leucine-rich repeat (eLRR) domains play an important role in plant immunity. However, the role of one class of eLRR plant proteins—the simple eLRR proteins—in plant defenses against herbivores remains largely unknown. Here, we found that a simple eLRR protein OsI-BAK1 in rice localizes to the plasma membrane. Its expression was induced by mechanical wounding, the infestation of gravid females of brown planthopper (BPH) Nilaparvata lugens or white-backed planthopper Sogatella furcifera and treatment with methyl jasmonate or abscisic acid. Silencing OsI-BAK1 (ir-ibak1) in rice enhanced the BPH-induced transcript levels of three defense-related WRKY genes (OsWRKY24, OsWRKY53 and OsWRKY70) but decreased the induced levels of ethylene. Bioassays revealed that the hatching rate was significantly lower in BPH eggs laid on ir-ibak1 plants than wild-type (WT) plants; moreover, gravid BPH females preferred to oviposit on WT plants over ir-ibak1 plants. The exogenous application of ethephon on ir-ibak1 plants eliminated the BPH oviposition preference between WT and ir-ibak1 plants but had no effect on the hatching rate of BPH eggs. These findings suggest that OsI-BAK1 acts as a negative modulator of defense responses in rice to BPH and that BPH might exploit this modulator for its own benefit.

The Arabidopsis MIK2 receptor elicits immunity by sensing a conserved signature from phytocytokines and microbes

Sessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that the Arabidopsis receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) from Brassicaceae plants as well as proteins present in fungal Fusarium spp. and bacterial Comamonadaceae, and elicits various immune responses. SCOOP signature peptides trigger immune responses and altered root development in a MIK2-dependent manner with a sub-nanomolar sensitivity. SCOOP12 directly binds to the extracellular leucine-rich repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identifies a plant receptor that bears a dual role in sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.

Molecular study of sticky platelet syndrome using exome sequencing

Introduction: Sticky platelet syndrome (SPS) is a prothrombotic condition characterized by increased platelet aggregation that causes arterial and venous thrombosis. Its diagnosis is reached by identifying increased aggregation using low concentrations of adenosine diphosphate and epinephrine in platelet aggregation tests. Objectives: To identify common mutations through exome sequencing in two patients from the same family diagnosed with SPS and, thus, contribute to the molecular study of this disease. Materials and methods: Descriptive study. In January 2018, exome sequencing was performed in a 10-year-old patient treated at Fundación HOMI (Bogotá D.C., Colombia), index case, and in one of his adult first-degree relatives, both with a history of thrombotic disease and diagnosed with SPS. Exome sequencing was performed at the Complexo Hospitalario Universitario de Santiago de Compostela (Spain) using the SureSelect Clinical Research Exome V2 software by Agilent. Results: Exome sequencing led to detect genetic variants in both cases when compared with the reference sequence. The following variant was identified in the two samples: a cytosine to thymine transition at position c.236 (NM_000174.4) of the glycoprotein (GP)Ib-IX-V complex platelet membrane receptor, which causes a heterozygous transition of the amino acid threonine to isoleucine (i.e., a transition from hydrophilic amino acid to a hydrophobic amino acid) at position p. 79 of the extracellular leucine-rich repeat domain of GPIbα subunit of the (GP)Ib–IX complex, involving a conformational change of the main receptor of ligands IB alpha, which might result in platelet hyperaggregation and thrombosis. This variant has not been described in patients with SPS to date. Conclusion: The mutation identified in both samples could be related to SPS considering the importance of glycoprotein IX in platelet function.

Toll-like receptors in dermatology, venereology, and leprosy

Toll-like receptors (TLRs) represent a family of Type I transmembrane proteins characterized by an extracellular leucine-rich repeat domain and a cytoplasmic domain. TLRs represent a conserved group of receptors which help the immune system to function properly. Different TLRs are associated with an array of skin diseases. TLR agonists and antagonists have great potential for the treatment of allergic and inflammatory diseases.

Constitutive Activation of Leucine-Rich Repeat Receptor Kinase Signaling Pathways by BAK1-INTERACTING RECEPTOR-LIKE KINASE3 Chimera.

Receptor kinases with extracellular leucine-rich repeat domains (LRR-RKs) form the largest group of membrane signaling proteins in plants. LRR-RKs can sense small molecule, peptide, or protein ligands and may be activated by ligand-induced interaction with a shape complementary SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor kinase. We have previously shown that SERKs can also form constitutive, ligand-independent complexes with the LRR ectodomains of BAK1-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3) receptor pseudokinases, negative regulators of LRR-RK signaling. Here, we report that receptor chimera in which the extracellular LRR domain of BIR3 is fused to the cytoplasmic kinase domains of the SERK-dependent LRR-RKs BRASSINOSTEROID INSENSITIVE1, HAESA and ERECTA form tight complexes with endogenous SERK coreceptors in the absence of ligand stimulus. Expression of these chimeras under the control of the endogenous promoter of the respective LRR-RK leads to strong gain-of-function brassinosteroid, floral abscission, and stomatal patterning phenotypes, respectively. Importantly, a BIR3-GASSHO1 (GSO1)/SCHENGEN3 (SGN3) chimera can partially complement sgn3 Casparian strip formation phenotypes, suggesting that SERK proteins also mediate GSO1/SGN3 receptor activation. Collectively, our protein engineering approach may be used to elucidate the physiological functions of orphan LRR-RKs and to identify their receptor activation mechanism in single transgenic lines.

Identificación de polimorfismos en el gen <i>RXAM1</i> de yuca y su asociación con la resistencia a la bacteriosis vascular

La yuca (Manihot esculenta Crantz) es un cultivo importante en regiones del trópico que proporciona alimento para cerca de 1000 millones de personas en todo el mundo. La enfermedad bacteriana más importante es la bacteriosis vascular causada por Xanthomonas axonopodis pv. manihotis (Xam). Recientemente se logró identificar un gen de resistencia denominado RXAM1, el cual codifica para una proteína que posee un dominio LRR (Leucine-Rich Repeat) extracelular y un dominio STK (Serine Threonine Kinase) citoplasmático. RXAM1 colocaliza con un QTL que explica el 13 % de la resistencia a la cepa CIO136 de Xam. En este trabajo se evaluó la respuesta a la infección con la cepa XamCIO136 en diez diferentes variedades de yuca lo cual permitió identificar que las variedades TMS60444, SG10735, MCOL1522, MCOL1505 y MCOL2215 fueron susceptibles, mientras que CM6438-14, CM523-7 y MBRA902 se comportaron como resistentes. Así mismo se identificaron polimorfismos tipo SNPs (Single Nucleotide Polymorphism) en el gen RXAM1 en el mismo grupo de variedades. Las variedades SG10735, CM6438-14, TMS6044 y MBRA685 presentaron el mayor nivel de polimorfismos, mientras que las variedades CM523-7, CM2177-2 y MCOL1522 fueron menos polimórficas para este gen. Los análisis estadísticos no permitieron identificar una asociación significativa entre el fenotipo y los polimorfismos identificados. Este estudio representa un primer esfuerzo con miras a asociar variantes alélicas con el fenotipo de respuesta a la bacteriosis vascular.

Molecular Characterization of Two Toll Receptors in Hyriopsis cumingii and Their Potential Roles in Antibacterial Response.

Tolls/Toll-like receptors (TLRs) play a key role in innate immunity by detecting the invading microbes and subsequently activating downstream signaling cascades. In this study, two new molluscan Toll members (designed as HcToll6 and HcToll7) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). The obtained HcToll6 full-length cDNA was 3207 bp consisting of a 2223 bp open reading frame (ORF) that encoded a peptide of 740 amino acids. HcToll7 cDNA is a 3216 bp molecule that contains an ORF of 2139 bp encoding a protein of 712 amino acids. The deduced HcToll6 and HcToll7 proteins share two common structures: extracellular leucine-rich repeat (LRR) domains and intracellular Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time PCR results showed that HcToll6 and HcToll7 were mainly expressed in the hepatopancreas and the gills, and they responded rapidly to bacterial stimulation. RNA interference by dsRNA results revealed that HcToll6 and HcToll7 RNAi strongly decreased the expression of lysozyme (HcLyso) and defensin (HcDef) in the gills of RNAi-treated mussels with Vibrio parahaemolyticus challenge. As a pattern recognition receptor, the prokaryotic expressed the recombinant LRR domains of HcToll6 and HcToll7 (rHcToll6-LRR and rHcToll7-LRR) could bind to Gram-positive and Gram-negative bacteria and had a strong tendency to recognize lipopolysaccharide (LPS) and peptidoglycan (PNG). rHcToll6-LRR and rHcToll7-LRR exhibited a significant in vitro bactericidal activity against V. parahaemolyticus and Staphylococcus aureus. These findings provide useful information to characterize Tolls in mussels.

Transcriptome-wide identification, functional characterization, and expression analysis of two novel invertebrate-type Toll-like receptors from disk abalone (Haliotis discus discus)

Toll-like receptors (TLRs) are well-known pattern recognition receptors that play key immunological roles in a diverse range of organisms. In this study, two novel invertebrate TLRs from disk abalone (designated as AbTLR-A and AbTLR-B) were identified and functionally characterized for the first time. AbTLR-A and AbTLR-B comprised the typical TLR domain architecture containing an extracellular leucine-rich repeat domain, transmembrane domain, and Toll/interleukin-1 receptor domain. Expressional analysis revealed that both TLRs were constitutively expressed at all the early embryonic stages of disk abalone analyzed, with the highest level of AbTLR-A found at the 16-cell stage and AbTLR-B at the trochophore stage. According to tissue distribution analysis, prominent mRNA expression of AbTLR-A and AbTLR-B was detected in the hemocytes and gills, respectively. AbTLR-A and AbTLR-B mRNAs were significantly up-regulated in response to Gram-negative Vibrio parahemolyticus, Gram-positive Listeria monocytogenes, and viral hemorrhagic septicemia virus injections in abalone hemocytes and gills. Overexpression of AbTLR-A and AbTLR-B in HEK293T cells directly activated nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) responsive reporters. Neither TLRs showed a high response to pathogen-associated molecular patterns in vitro. Co-expression of AbTLR-A and AbTLR-B with AbMyD88-2 and AbMyD88-X activated NF-κB-responsive reporters in a synergetic manner. These findings demonstrate the involvement of AbTLR-A and AbTLR-B in abalone innate immunity.

SpToll1 and SpToll2 modulate the expression of antimicrobial peptides in Scylla paramamosain

Tolls and Toll-like receptors (TLRs) were the first pattern recognition receptors (PRRs) identified to play key roles in host innate immunity. However, relatively little is known about other types of Toll-like receptors in Scylla paramamosain, although a Toll-like receptor (SpToll1) has recently been cloned. In this study, we cloned and characterized another novel Toll-like receptor 2 (SpToll2) from S. paramamosain. The full-length cDNA of SpToll2 is 3391 bp with a 2646 bp open reading frame (ORF) encoding a putative protein of 881 amino acids, and predicted to contain six extracellular leucine-rich repeat (LRR) domains, a transmembrane domain and an intracellular Toll/IL-1 receptor (TIR) domain. Phylogenetic analysis revealed that SpToll2 clustered with Drosophila Toll1, and shared high homology with PtToll4. Real-time qPCR analysis showed that SpToll2 was widely expressed in all tissues tested, with the highest level found in hemocytes and hepatopancreas while the lowest in heart and muscle. The transcript levels of both SpToll1 and SpToll2 in mud crabs hemocytes was induced following challenge with Vibrio parahaemolyticus, Staphylococcus aureus, Polyinosinic: polycytidylic acid (Poly I:C) and white spot syndrome virus (WSSV). In addition, recombinant SpToll1-LRR and SpToll2-LRR proteins could bind to V. parahaemolyticus, S. aureus, Escherichia coli, and Beta Streptococcus. In order to study the signaling pathway of AMPs' expression in mud crab, RNA interference were used to test the expression of SpAMPs after the challenges with V. parahaemolyticus or S. aureus. The data suggested that SpToll1and SpToll2 could regulate the transcripts of several AMPs and four immune related mediators (SpMyD88, SpTube, SpPelle and SpTRAF6) at different scale. While silencing of SpToll1 post pathogens challenge attenuated the expression of SpHistin, SpALF1 and SpALF5 in mud crab's hemocytes, depletion of SpToll2 post pathogens challenge inhibited the expression of SpALF1-6, SpGRP, SpArasin and SpHyastastin. Furthermore, the results of overexpression assay also showed SpToll1 and SpToll2 could enhance the promoter activities of SpALFs in mud crab. Taken together, these results indicated that SpToll1 and SpToll2 might play important roles in host defense against pathogen invasions in S. paramamosain.

Domain Swap Approach Reveals the Critical Roles of Different Domains of SYMRK in Root Nodule Symbiosis in Lotus japonicus.

Symbiosis receptor kinase (SYMRK) is a cell membrane-localized protein kinase containing extracellular malectin-like domain (MLD) and leucine-rich repeat (LRR) domains, which is critically required for both root nodule symbiosis (RNS) and arbuscular mycorrhizal symbiosis (AMS). SYMRK is widely distributed in the genomes of different plant species; however, the contribution of different domains of SYMRK and its homologs from other plant species to RNS is largely unclear. In this study, SYMRK and its homologs from three typical plant species including Medicago truncatula (for both RNS and AMS), Oryza sativa (for AMS but not RNS), and Arabidopsis thaliana (for neither RNS or AMS) were investigated using domain swap approach in response to rhizobia in Lotus japonicus. Full-length SYMRK from rice and Medicago but not from Arabidopsis could complement Lotus symrk-409 mutant plants to contribute RNS. The chimeric protein with the extracellular domain (ED) of LjSYMRK and cytoplasmic domains (CD) of SYMRK from both Medicago and rice but not Arabidopsis could contribute to RNS in Lotus, suggesting that the CD of SYMRK is required for symbiotic signaling. The chimeric receptors containing the CD of LjSYMRK (SYMRKCD) and the EDs of MtDMI2 (MtDMI2ED), OsSYMRK (OsSYMRKED), AtSYMRK (AtSYMRKED), NFR1 (NFR1ED), and NFR5 (NFR5ED) could complement Lotus symrk-409 mutant plants to develop nodules. However, MtDMI2 could partially complement Lotus symrk-409 mutants to form both effective nodules and ineffective bumps, which is similar to the complementation results from MtDMI2ED-LjSYMRKCD and LjSYMRKGDLC in Lotus symrk-409 mutants, suggesting that ED of SYMRK has a very fine-tune regulation for RNS in Lotus. The deletion of either MLD or LRR on SYMRKGDLC (a mutant version of SYMRK with GDPC motif replaced by GDLC) could contribute to RNS when overexpressed in Lotus symrk-409 mutants, suggesting that MLD and LRR domains might work together to be involved in symbiotic signaling and the LRR domain might play a negative role in LjSYMRKGDLC-mediated RNS. By mutagenizing the conserved amino acids on LRR domain, five serine residues were found to be required for the function of LjSYMRKGDLC in RNS. These finding precisely refine the molecular mechanisms of SYMRK function in symbiotic signaling in L. japonicus.

Molecular cloning of sea perch (Lateolabrax japonicus ) TLR1 and analysis of its expression pattern after stimulation with various bacteria

Toll-like receptors (TLRs) play an indispensable role in fish immunity, being involved in pathogen recognition and the triggering of immune reactions. Here, a member of the TLR family, TLR1, from Lateolabrax japonicus was characterized and its expression pattern and intracellular localization were analysed. The full-length LjTLR1 cDNA (2,755 bp) was found to encode a polypeptide of 827 amino acids. The deduced amino acid sequence contained three main structural domains: an extracellular leucine-rich repeat domain, a transmembrane domain and a Toll/IL-1 receptor domain. Tissue distribution analysis indicated that LjTLR1 was expressed in all of the examined tissues to varying degrees, with the highest levels being measured in the head kidney. In order to assess the antibacterial functions of LjTLR1 during infection, the pathogenic bacteria Vibrio harveyi and Streptococcus agalactiae were used. LjTLR1 was significantly upregulated in the three immune organs (the head kidney, spleen and liver) following bacterial stimulation, and its expression was detected 6 hr after initial exposure. In mRNA in situ hybridization experiments, positive signals were more numerous in the treatment group than the control group, verifying the expression patterns observed. Assessment of the intracellular localization of LjTLR1 revealed it to be present in the cytoplasm. These results indicate the potential role of LjTLR1 in immune responses to bacterial infection. This study enriches our knowledge of L. japonicus immune genes and provides a theoretical basis for further research concerning the antibacterial functions of fish TLRs during infection.

Goose toll-like receptor 3 (TLR3) mediated IFN-γ and IL-6 in anti-H5N1 avian influenza virus response

Induction of the innate immune pathways is critical for early anti-viral defense. How geese recognize viral molecules and activate these pathways is not well understood. In mammals, Toll-like receptor 3 (TLR3) recognizes double-stranded RNA. Activation of TLR3 induces the activation of NF-кB and the production of type-I interferon. In this study, the goose TLR3 gene was cloned using rapid amplification of cDNA ends. Goose TLR3 encoded an 896-amino-acid protein, containing a signal secretion peptide, 14 extracellular leucine-rich repeat domains, a transmembrane domain, a Toll/interleukin-1 receptor signaling domain, and shared 46.7–84.4% homology with other species. Tissue expression of goose TLR3 varied markedly and was highest in the pancreas and lowest in the skin. Human embryonic kidney 293 cells transfected with goose TLR3 and NF-κB-luciferase-containing plasmids responded significantly to poly i:c. The expression of TLR3, IL-6 and IFN-γ mRNA, but not IL-1 mRNA, was significantly upregulated after poly i:c or high pathogenic avian influenza virus (H5N1) stimulation in goose peripheral blood mononuclear cells cultured in vitro. Furthermore, geese infected with H5N1 showed significant upregulation of TLR3, especially in the lung and brain. We conclude that goose TLR3 is a functional TLR3 homologue of the protein in other species and plays an important role in virus recognition.

Leucine-rich repeat receptor-like gene screen reveals that Nicotiana RXEG1 regulates glycoside hydrolase 12 MAMP detection

Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.

Molecular characterization and expression analysis of toll-like receptors 5 and 22 from natural triploid Carassius auratus

Innate immunity, as the most primitive and universal host defense in fish, constitutes an efficient first line of defense to combat invading microbes. Toll-like receptors (TLRs) play essential roles in the innate immunity, and TLR5 and TLR22 are two important TLRs that can recognize flagellin and double stranded RNA (dsRNA), respectively. In this study, we identified and characterized two TLRs genes of Qihe crucian carp (Carassius auratus) (designated as CaTLR5 and CaTLR22). The full-length cDNA sequence of CaTLR5 was cloned with 2972 bp including a 140 bp 5’-terminal untranslated region (UTR), a 183 bp 3′-UTR, and a 2649 bp open reading frame (ORF) encoding a deduced protein with 882 amino acids. The full-length cDNA of CaTLR22 was identified to be 3613 bp, consisting of a 228 bp 5′-UTR, a 547 bp 3′-UTR, and a 2838 bp ORF encoding a predicted protein of 945 amino acids. A typical TLR structure (an extracellular leucine-rich repeat domain, a transmembrane domain, and an intracellular Toll/IL-1 receptor domain) was found in CaTLR5 and CaTLR22. For either CaTLR5 or CaTLR22 gene, the mRNA expression levels varied in the different periods during the early stages of development. It was suggested that expression changes of gene CaTLR5 and CaTLR22 at mRNA levels were involved in developmental regulation in the early stages, and it was postulated that CaTLR5 and CaTLR22 play the important roles in immune defense in the early development stages of fish. Quantitative Real-Time PCR (qRT-PCR) revealed that CaTLR5 and CaTLR22 were constitutively expressed in all eleven tissues examined, although the mRNA expression level varied considerably among the different tissues. Following exposure to polyI:C, flagellin, and Aeromonas hydrophila, CaTLR5 and CaTLR22 were up-regulated in different tissues, and it was suggested that CaTLR5 and CaTLR22 were involved in the immune response of Qihe crucian carp against pathogenic invasions. The present findings will provide the valuable information for understanding the structure, function, expression, and the immune defense process of CaTLR5 and CaTLR22 in Qihe crucian carp, and provide new insights for developing the new strategies of disease control to protect fish against pathogens infection.

Analysis for Protein Glycosylation of Pattern Recognition Receptors in Plants.

Recognition of molecules typical of microbes or aberrant cellular states, termed microbe- or danger-associated molecular patterns (MAMPs/DAMPs), respectively, provides an important step in plant and animal innate immunity. In plants, pattern recognition receptors (PRRs) identified to date are limited to membrane-associated proteins, of which the majority has an extracellular leucine-rich repeat (LRR) or lysine-motif (LysM) domain. These PRRs undergo quality control (QC) in the Endoplasmic Reticulum (ER) that is dependent on Asn (N)-linked glycosylation (Glc3Man9GlcNAc2 conjugation) of their extracellular domain. In Arabidopsis, genetic studies have revealed that a subset of these PRRs require an intact N-glycosylation pathway in the ER for their biogenesis and function. Here, we describe methods for immunoblot-based detection of protein glycosylation states in plants.

Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 signaling.

Receptor kinases convey diverse environmental and developmental inputs by sensing extracellular ligands. In plants, one group of receptor-like kinases (RLKs) is characterized by extracellular leucine-rich repeat (LRR) domains, which interact with various ligands that include the plant hormone brassinosteroid and peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) type. For instance, the CLE45 peptide requires the LRR-RLK BARELY ANY MERISTEM 3 (BAM3) to prevent protophloem formation in Arabidopsis root meristems. Here, we show that other proposed CLE45 receptors, the two redundantly acting LRR-RLKs STERILITY-REGULATING KINASE MEMBER 1 (SKM1) and SKM2 (which perceive CLE45 in the context of pollen tube elongation), cannot substitute for BAM3 in the root. Moreover, we identify MEMBRANE-ASSOCIATED KINASE REGULATOR 5 (MAKR5) as a post-transcriptionally regulated amplifier of the CLE45 signal that acts downstream of BAM3. MAKR5 belongs to a small protein family whose prototypical member, BRI1 KINASE INHIBITOR 1, is an essentially negative regulator of brassinosteroid signaling. By contrast, MAKR5 is a positive effector of CLE45 signaling, revealing an unexpected diversity in the conceptual roles of MAKR genes in different signaling pathways.

Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis.

Directional control of tip-growing cells is essential for proper tissue organization and cell-to-cell communication in animals and plants. In the sexual reproduction of flowering plants, the tip growth of the male gametophyte, the pollen tube, is precisely guided by female cues to achieve fertilization. Several female-secreted peptides have recently been identified as species-specific attractants that directly control the direction of pollen tube growth. However, the method by which pollen tubes precisely and promptly respond to the guidance signal from their own species is unknown. Here we show that tip-localized pollen-specific receptor-like kinase 6 (PRK6) with an extracellular leucine-rich repeat domain is an essential receptor for sensing of the LURE1 attractant peptide in Arabidopsis thaliana under semi-in-vivo conditions, and is important for ovule targeting in the pistil. PRK6 interacted with pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which are important for pollen tube growth through activation of the signalling switch Rho GTPase ROP1 (refs 7, 8). PRK6 conferred responsiveness to AtLURE1 in pollen tubes of the related species Capsella rubella. Furthermore, our genetic and physiological data suggest that PRK6 signalling through ROPGEFs and sensing of AtLURE1 are achieved in cooperation with the other PRK family receptors, PRK1, PRK3 and PRK8. Notably, the tip-focused PRK6 accumulated asymmetrically towards an external AtLURE1 source before reorientation of pollen tube tip growth. These results demonstrate that PRK6 acts as a key membrane receptor for external AtLURE1 attractants, and recruits the core tip-growth machinery, including ROP signalling proteins. This work provides insights into the orchestration of efficient pollen tube growth and species-specific pollen tube attraction by multiple receptors during male-female communication.