TIR Region Research Articles

Molecular characterization and comparisons of potato wart (Synchytrium endobioticum) in historic collections to recent findings in Canada and the Netherlands

AbstractSynchytrium endobioticum (Schilb.) Perc. is a chytrid fungus causing potato wart disease and is one of the most important quarantine diseases on cultivated potato. Infected host tissues develop warts rendering the crop unmarketable. Resting spores, that can remain viable and infectious for decades, are formed in warted tissues and are released into the surrounding soil when host tissue decays.To better understand the pathogen’s diversity and to potentially uncover pathways of migrations and introduction events, molecular characterization was performed on the historical S. endobioticum resting spore collection of the Dutch National Plant Protection Organization. Mitochondrial genomes were assembled and annotated, and four novel structural variants were identified from these materials with intronic presence-absence variation in cox1 or cob genes and structural variation in the dpoB – TIR region. Several fungal isolates were shown to contain mixtures of structural variants. We analyzed the mitogenomic sequences obtained from recent potato wart disease findings in Canada and the Netherlands in the context of the historical materials and found that fungal isolates from the new Dutch outbreak contained a specific mixture of mitogenomic variants previously not observed in the Netherlands. Based on the mitogenomic profile, pathotype 38(Nevşehir) was suspected which was later verified with the Spieckermann bioassay. To further facilitate dissemination of data and interactive visual analytics we created a public Nextstrain webpage with S. endobioticum mitogenomic sequences and associated metadata on their geographic origin, pathotype identity and (mixture) of mitogenomic variants (https://nextstrain.nrcnvwa.nl/Sendo).

Development of candidate gene-based markers and map-based cloning of a dominant rust resistance gene in cultivated groundnut (Arachis hypogaea L.)

A dominant rust resistance gene, VG 9514-Rgene was isolated through map-based cloning. Sequence analysis revealed non-synonymous mutations in the TIR, NBS and LRR region of the R-protein. Candidate gene-based markers from these SNPs revealed complete co-segregation of the isolated VG 9514-Rgene with rust resistance in a RIL population and confirmed their map position in between FRS 72 and SSR_GO340445 markers in arahy03 chromosome. Blastp search of VG 9514-Rprotein detected Arahy.T6DCA5 with >80.0% identity that localized at 142,544,745.0.142,549,184 in arahy03 chromosome. Ka/Ks calculation revealed that VG 9514-Rgene had undergone positive selection compared to four homologous genes in the groundnut genome. Homology based structure modelling of this R-protein revealed a typical consensus three-dimensional folding of TIR-NBS-LRR protein. Non-synonymous mutations in susceptible version of R-protein were mapped and found E268Q mutation in hhGRExE motif, Y309F in RNBS-A motif and I579T in MHD motif of NB-ARC domain are probable candidates for loss of function.

Genetic diversity of the toll-like receptor 2 (TLR2) in hare (Lepus capensis) populations from Tunisia

Toll-like receptors (TLRs) are a major group of proteins that recognize molecular components of infectious agents, known as pathogen associated molecular patterns (PAMPs). The structure of these genes is similar and characterized by the presence of an ectodomain, a signal transmembrane segment and a highly conserved cytoplasmic domain. The latter domain is homologous to the human interleukin-1 receptor (IL1R) and human IL-18 receptor (IL-18R) and designated TIR domain. The latter domain of the TLR genes was suggested to be very conservative and its evolution is driven by purifying selection. Variability and evolution of the TIR sequences of TLR2 gene were studied in three hare populations from Tunisia with different ecological characteristics (NT–North Tunisia with Mediterranean, CT–Central Tunisia with semi-arid, and ST–South Tunisia with arid climate). Sequencing of a 372bp fragment of TIR2 revealed 25 alleles among 110 hares. Twenty variable nucleotide positions were detected, of which 7 were non-synonymous. The highest variability was observed in CT, with 16 polymorphic positions. In ST, only 4 polymorphic nucleotide positions were detected with all diversity values lower than those recorded for the other two populations. By using several approaches, no positive selection was detected. However, evidence of purifying selection was found at two positions. The logistic models of the most common TIR2 protein variant that we run to examine whether its occurrence was affected by climatic variation independent of the geographic sample location suggested only a longitudinal effect. Finally, the mapping of the non-synonymous mutations to the inferred tertiary protein structure showed that they were all localized in the different loop regions. Among all non-synonymous substitutions, three were suggested to be deleterious as evidenced by PROVEAN analysis. The observed patterns of variability characterized by low genetic diversity in ST might suggest that the TIR region was more affected, than other markers, by genetic drift or/and that these patterns were shaped by different selective pressures under different ecological conditions. Notably, this low diversity was not detected by other (putatively neutral) microsatellite markers analysed in the course of other studies. But low diversity was also found for two MHC class II adaptive immune genes. As expected from functionally important regions, the evolution of the TIR2 domain is mainly driven by purifying selection. However, the occurrence of deleterious non-synonymous substitutions might highlight the flexible evolution of the TIR genes and/or their interactions with other proteins.

Retrieving vegetation canopy water content from hyperspectral thermal measurements

The retrieval of vegetation canopy water content using thermal hyperspectral (TIR, 8–14μm) measurements is investigated in this study. Vegetation water content indicators such as fuel moisture content (FMC, %, mass-based) and equivalent water thickness (EWT, gcm−2, area based) play significant roles in plant physiology, as well as in the modelling of fire risk and behavior, particularly in forests. Although retrieval of these parameters, in particular EWT, has been demonstrated from optical and TIR measurements, to our knowledge their prediction at canopy level in the thermal part of the electromagnetic spectrum has not yet been investigated. Therefore, the application of hyperspectral TIR data for predicting FMC and EWT parameters at canopy level is explored here. The emissivity of spectral data in the TIR region is measured for four species (Azalea japonica, Buxus sempervirens, Euonymus japonicus, and Ficus benjamina) under controlled laboratory conditions, using a portable MIDAC Fourier transform infrared spectrometer. EWT, FMC, and their corresponding canopy emissivity measurements are assessed by destructive sampling of the leaves. Leaf area, as well as fresh and dry mass of the harvested leaves, is determined for all four species. Partial least square regression and artificial neural networks, using various spectral subsets, are used to predict the two variables of interest. Higher estimation accuracies have been obtained for both FMC and EWT at canopy level using artificial neural networks. Unexpectedly, the FMC at canopy level, as a mass-based variable, more accurately retrieved using either method. This is contrary to previous findings using multispectral and hyperspectral data. Our results suggest that plant mass may play a greater role in determining spectral emissivity than plant area does.

Enteropathogenic Escherichia coli Tir recruits cellular SHP-2 through ITIM motifs to suppress host immune response

Immune responses to pathogens are regulated by immune receptors containing either an immunoreceptor tyrosine-based activation motif (ITAM) or an immunoreceptor tyrosine-based inhibitory motif (ITIM). The important diarrheal pathogen enteropathogenic Escherichia coli (EPEC) require delivery and insertion of the bacterial translocated intimin receptor (Tir) into the host plasma membrane for pedestal formation. The C-terminal region of Tir, encompassing Y483 and Y511, shares sequence similarity with cellular ITIMs. Here, we show that EPEC Tir suppresses the production of inflammatory cytokines by recruitment of SHP-2 and subsequent deubiquitination of TRAF6 in an ITIM dependent manner. Our findings revealed a novel mechanism by which the EPEC utilize its ITIM motifs to suppress and evade the host innate immune response, which could lead to the development of novel therapeutics to prevent bacterial infection.

In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

BackgroundIn silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing). The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions.ResultsWe designed synthetic genes encoding the carboxy-terminal fragment of Intimin, the middle region of Tir and the carboxy-terminal part of EspA. These multi genes were synthesized with codon optimization for a plant host and were fused together by the application of four repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted.Conclusiona structural model for a chimeric gene from LEE antigenic determinants of EHEC is presented. It may define accessibility, solubility and immunogenecity.

Using IRS Products to Recover 7ETM+ Defective Images

On May 31st, 2003, Landsat 7 faced an anomaly in the Scan Line Corrector (SLC) normal operation. This malfunctioning of SLC caused the individual scan lines alternately overlap each other and consequently produce large gaps at the edges of the image. Regarding the unique specification of ETM+ sensor on board of Landsat-7 satellite such as its spectral bands in the shortwave IR and TIR region and its suitable spatial resolution which is ideal for most of the scientific researches, a technique for the reconstruction of the defected images due to the SLC malfunctioning, was built up. Due to the availability of IRS/1D LISS-III images for our region of interest i.e., southwest of Iran, it was decided to use these images to recover 7ETM+ products. The procedure in reconstructing the defected 7ETM+ images is divided in to two stages. In the first stage, after implementation of some preprocessing to both LISS-III and 7ETM+, a linear regression model between bands 3 and 4 of 7ETM+ and bands 2 and 3 of LISS-III was setup. This model is used to fill up the missing places in 7ETM+ defected image and produced two new images in bands 3 and 4. In the second stage, these two newly reconstructed images of 7ETM+ were used to recover images of 7ETM+ in other spectral bands. At this stage, two methods were introduced, one using linear relationship between band 3 and bands 1 and 2 and in the second method we introduced a planar relationship between bands 3 and 4 with each one of bands 5, 6 and 7. The models are applied to few images and are found to be fairly reliable. The primary and necessary conditions for applying these methods have been explained in detail.

Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspFU-mediated actin assembly and harbours N-terminal sequences that influence pedestal length

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into 'pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspF(U). To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspF(U) and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function.

Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defense responses.

Tobacco was transformed with three different alleles (L2, L6, and L10) of the flax rust resistance gene L, a member of the toll interleukin-1 receptor, nucleotide-binding site, leucine-rich repeat (TIR-NBS-LRR) class of plant disease resistance genes. L6 transgenics had a stunted phenotype, expressed several defense response genes constitutively, and had increased resistance to the fungus Cercospora nicotianae and the oomycete Phytophthora parasitica pv. nicotianae. L2 and L10 transgenics, with one exception for L10, did not express these phenotypes, indicating that the activation of tobacco defense responses is L6 allele-specific. The phenotype of the exceptional L10 transgenic plant was associated with the presence of a truncated L10 gene resulting from an aberrant T-DNA integration. The truncated gene consisted of the promoter, the complete TIR region, and 39 codons of the NBS domain fused inframe to a tobacco retrotransposon-like sequence. A similar truncated L10 gene, constructed in vitro, was transiently expressed in tobacco leaves and gave rise to a strong localized necrotic reaction. Together, these results suggest that defense signaling properties of resistance genes can be expressed in an allele-specific and pathogen-independent manner when transferred between plant genera.

Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination.

Multiple alleles controlling different gene-for-gene flax rust resistance specificities occur at the L locus of flax. At least three distinct regions can be recognized in the predicted protein products: the Toll/interleukin-1 receptor homology (TIR) region, a nucleotide binding site (NBS) region, and a leucine-rich repeat (LRR) region. Replacement of the TIR-encoding region of the L6 allele with the corresponding regions of L2 or LH by recombination changed the specificity of the allele from L6 to L7. Replacement of the TIR and most of the NBS-encoding region of L10 with the equivalent region of L2 or L9 generated recombinant alleles having a novel specificity. However, replacement of the L10 TIR-encoding region with the TIR-encoding region of L2 gave rise to an allele with no detectable specificity. These data indicate that non-LRR regions can determine specificity differences between allelic gene products and that functional specificity involves interactions between coadapted polymorphic regions in the protein products of the alleles. Evidence for the action of diversifying selection on the TIR region is observed.

Human Colostrum and Serum Contain Antibodies Reactive to the Intimin-Binding Region of the Enteropathogenic Escherichia coli Translocated Intimin Receptor

In Brazil, enteropathogenic Escherichia coli (EPEC) diarrhoea is endemic in young infants. A characteristic feature of EPEC adhesion to host cells is intimate attachment leading to the formation of distinctive "attaching and effacing" (A/E) lesions on mammalian cells. Two genes directly involved in intimate adhesion, eae and tir, encode the adhesion molecule intimin and its translocated receptor Tir, respectively. The intimin-binding domain of Tir was recently mapped to the middle part of the polypeptide (Tir-M), and the amino (Tir-N) and carboxy (Tir-C) termini were found to be located within infected host cells. Recently, it was shown that colostrum samples from mothers living in Sao Paulo contain IgA-class antibodies reactive with a number of proteins associated with EPEC virulence. It has also been shown that patients infected with verocytotoxin-producing E. coli O157 can produce antibodies to Tir. In the current study antibody responses to the different Tir domains were analyzed in sera and colostrum samples collected in an EPEC-endemic area of Brazil. Recombinant Tir, Tir-N, Tir-M, and Tir-C were expressed as His-tagged protein in E. coli BL21a and purified on nickel columns. Western blot analysis was used to investigate colostrum IgA- and serum IgG-class antibodies reactive with the Tir fragments. Anti-Tir IgG antibodies were detected in the serum of children, with (63%) or without (50%) diarrhoea. Anti-Tir IgA-class antibodies were detected in all the colostrum pools tested. With the use of both serum IgG- and colostrum IgA-class antibodies, an immunodominant domain of the Tir-polypeptide, Tir M, was identified. The intimin-binding region of Tir (Tir-M) is the immunodominant region of the polypeptide in humans. Both serum IgG-class and colostrum IgA-class antibodies reacted predominantly with the Tir-M domain.

Antibody response of patients infected with verocytotoxin-producing Escherichia coli to protein antigens encoded on the LEE locus

Sera from patients infected with verocytotoxin-producing Escherichia coli (VTEC) O157, from patients with antibodies to E. coli O157 lipopolysaccharide (LPS) and from healthy controls were examined for antibodies to proteins involved in expressing the attaching and effacing phenotype. After SDS-PAGE, purified recombinant intimin, EspA-filament structural protein, translocated protein EspB and three separate domains of the translocated intimin receptor (Tir) were tested for reaction with patients' sera by immunoblotting. An ELISA was also used to detect antibodies to intimin in sera from E. coli O157 LPS antibody-positive individuals. Seven of nine culture-positive patients and one control patient had antibodies to EspA. Five of these patients and two controls had serum antibodies to the intimin-binding region of Tir, whereas none of the sera contained antibodies binding to either of the intracellular domains of Tir. By immunoblotting, 10 of 14 culture-positive patients had antibodies to the conserved region of intimin, eight of whom were infected with E. coli O157 phage type 2. Thirty-six of 60 sera from culture-negative but E. coli O157 LPS antibody-positive patients had antibodies to intimin as determined by ELISA. The secreted proteins are expressed in vivo during infection and are considered as pathogenic markers. Antibodies to these proteins may form the basis of a serodiagnostic test for the detection of patients infected with VTEC which carry the locus for the enterocyte effacement pathogenicity island and provide an adjunct test to the established serological tests based on VTEC LPS.

Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity.

Thirteen alleles (L, L1 to L11, and LH) from the flax L locus, which encode Toll/interleukin-1 receptor homology-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) rust resistance proteins, were sequenced and compared to provide insight into their evolution and into the determinants of gene-for-gene resistance specificity. The predicted L6 and L11 proteins differ solely in the LRR region, whereas L6 and L7 differ solely in the TIR region. Thus, specificity differences between alleles can be determined by both the LRR and TIR regions. Functional analysis in transgenic plants of recombinant alleles constructed in vitro provided further information: L10-L2 and L6-L2 recombinants, encoding the LRR of L2, conferred L2 resistance specificity, and an L2-L10 recombinant, encoding the LRR of L10, conferred a novel specificity. The sequence comparisons also indicate that the evolution of L alleles has probably involved reassortment of variation, resulting from accumulated point mutations, by intragenic recombination. In addition, large deletion events have occurred in the LRR-encoding regions of L1 and L8, and duplication events have occurred in the LRR-encoding region of L2.

A novel integrated-twin-guide (ITG) optical switch with a built-in TIR region

The authors propose a novel integrated-twin-guide (ITG) optical switch with a built-in TIR region to enlarge the spatial separation of output lights, which is suitable for a compact optical switching matrix array. A switching operation was observed in a GaInAsP/InP MQW ITG optical switch utilizing electric-field induced refractive index variation. >