Characterization,expression analysis and localization pattern of toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella

In this study, the toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up‐regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells.     

Characterizations of four toll‐like receptor 4s in grass carp Ctenopharyngodon idellus and their response to grass carp reovirus infection and lipopolysaccharide stimulation

In this study, the subcellular localization, tissue distribution and response to grass carp reovirus (GCRV) infection and lipopolysaccharide (LPS) stimulation of four grass carp Ctenopharyngodon idellus toll‐like receptor 4 (tlr4) genes were investigated. All four genes were constitutively expressed in all tissues studied, but the subcellular localization and tissue exhibiting the highest expression differed for each protein. Following GCRV infection, all the four tlr4s were upregulated in all tissues examined, and stimulation of C. idellus kidney (CIK) cells with LPS resulted in downregulation of all four tlr4s. These results provide a foundation for further investigation of tlr4 genes in bony fishes.     

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease‐resistance proteins with nucleotide binding site (NBS) and leucine‐rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS‐LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS‐LRR disease‐resistance proteins and triggers the production of trans‐acting (ta)‐siRNAs, most of which target mRNAs of defense‐related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e‐derived ta‐siRNA‐mediated silencing on NBS‐LRR‐disease‐resistance proteins. It is speculated that a miR482‐mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.     

Molecular cloning and expression analysis of myd88 from oriental weatherfish (Misgurnus anguillicaudatus) in response to bacterial challenge

Myeloid differentiation factor 88 (Myd88) plays an important role in both innate and adaptive immune response. In this study, the full-length complementary DNA (cDNA) of myd88 from Misgurnus anguillicaudatus was characterized. The myd88 cDNA is 1333 bp in length and contains an 855 bp open reading frame encoding a predicted protein of 284 amino acids. The predicted protein possesses typical Myd88 domain structural features including a death domain in the N-terminus, and box 1, 2, and 3 motifs of the Toll/IL-1 receptor domain in the C-terminus. Quantitative real-time PCR (qRT-PCR) revealed that myd88 messenger RNA (mRNA) was ubiquitously expressed in all examined tissues, especially highly in brain, kidney, blood, intestines and liver. qRT-PCR and western blotting were used to determine the mRNA and protein levels of Myd88 after Aeromonas veronii challenge, respectively. The Myd88 was remarkably upregulated in response to infection of A. veronii. These results suggested that Myd88 may play a vital role during the immune response of M. anguillicaudatus against bacterial infection.     

Bcl10 synergistically links CEACAM3 and TLR‐dependent inflammatory signalling

The neutrophil‐specific innate immune receptor CEACAM3 functions as a decoy to capture Gram‐negative pathogens, such as Neisseria gonorrhoeae, that exploit CEACAM family members to adhere to the epithelium. Bacterial binding to CEACAM3 results in their efficient engulfment and triggers activation of an nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB)‐dependent inflammatory response by human neutrophils. Herein, we report that CEACAM3 cross‐linking is not sufficient for induction of cytokine production and show that the inflammatory response induced by Neisseria gonorrhoeae infection is elicited by an integration of signals from CEACAM3 and toll‐like receptors. Using neutrophils from a human CEACAM‐expressing mouse line (CEABAC), we use a genetic approach to reveal a molecular bifurcation of the CEACAM3‐mediated antimicrobial and inflammatory responses. Ex vivo experiments with CEABAC‐Rac2^?/?, CEABAC‐Bcl10^?/?, and CEABAC‐Malt1^?/? neutrophils indicate that these effectors are not necessary for gonococcal engulfment, yet all 3 effectors contribute to CEACAM3‐mediated cytokine production. Interestingly, although Bcl10 and Malt1 are often inextricably linked, Bcl10 enabled synergy between toll‐like receptor 4 and CEACAM3, whereas Malt1 did not. Together, these findings reveal an integration of the specific innate immune receptor CEACAM3 into the network of more conventional pattern recognition receptors, providing a mechanism by which the innate immune system can unleash its response to a relentless pathogen.     

Interferon‐α curbs production of interleukin‐22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi

Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)‐α, either endogenously produced after exposure of cells to toll‐like receptor‐9‐activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti‐bacterial interleukin (IL)‐1/IL‐22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN‐α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection.     

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES‐2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose‐mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD‐like tlr0903 (xcsB) and endoglucanase‐like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose‐mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.     

Pathogenesis‐related protein 4b interacts with leucine‐rich repeat protein 1 to suppress PR4b‐triggered cell death and defense response in pepper

To control defense and cell‐death signaling, plants contain an abundance of pathogen recognition receptors such as leucine‐rich repeat (LRR) proteins. Here we show that pepper (Capsicum annuum) LRR1 interacts with the pepper pathogenesis‐related (PR) protein 4b, PR4b, in yeast and in planta. PR4b is synthesized in the endoplasmic reticulum, interacts with LRR1 in the plasma membrane, and is secreted to the apoplast via the plasma membrane. Binding of PR4b to LRR1 requires the chitin‐binding domain of PR4b. Purified PR4b protein inhibits spore germination and mycelial growth of plant fungal pathogens. Transient expression of PR4b triggers hypersensitive cell death. This cell death is compromised by co‐expression of LRR1 as a negative regulator in Nicotiana benthamiana leaves. LRR1/PR4b silencing in pepper and PR4b over‐expression in Arabidopsis thaliana demonstrated that LRR1 and PR4b are necessary for defense responses to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis (Hpa) infection. The mutant of the PR4b Arabidopsis ortholog, pr4, showed enhanced susceptibility to Hpa infection. Together, our results suggest that PR4b functions as a positive modulator of plant cell death and defense responses. However, the activity of PR4b is suppressed by interaction with LRR1.     

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.     

The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

The evolutionary characteristics and structural biology of Gallus toll‐like receptor 21

Toll‐like receptors (TLRs) are an important part of the innate immune system, acting as a first line of defense against many invading pathogens. The ligand known to bind Gallus toll‐like receptor 21 (gTLR21) is the unmethylated cytosine phosphate guanine dideoxy nucleotide motif; however, the evolutionary characteristics and structural biology of gTLR21 are poorly elaborated. Our results suggest that gTLR21 is phylogenetically and evolutionarily related to the TLR11 family and is perhaps a close ortholog of the Mus TLR13. Structural biology of homology modeling of the gTLR21 ectodomain structure suggests that it has no Z‐loop like that seen in Mus TLR9. The cytosolic toll‐IL‐1 receptor region of gTLR21 contains a central 4‐stranded parallel β‐sheet (βA‐βD) surrounded by 5 α‐helices (αA‐αE) on both sides, a highly conserved structure also seen in other TLRs. Molecular docking analysis reveals that the gTLR21 ectodomain has the potential to distinguish between different ligands. Homodimer analysis results also suggest that Phe842 and Pro844 of the BB loop and Cys876 of the αC helix in gTLR21 are conserved in other cytosolic toll‐IL‐1 receptor domains of other TLRs and may contribute to the docking of homodimers. Our study on the evolutionary characteristics and structural biology of gTLR21 reveals that the molecule may have a broader role to play in innate immune system; however, further experimental validation is required to confirm our findings.