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1.  Effect of Fungal Colonization of Wheat Grains with Fusarium spp. on Food Choice, Weight Gain and Mortality of Meal Beetle Larvae (Tenebrio molitor) 
PLoS ONE  2014;9(6):e100112.
Species of Fusarium have significant agro-economical and human health-related impact by infecting diverse crop plants and synthesizing diverse mycotoxins. Here, we investigated interactions of grain-feeding Tenebrio molitor larvae with four grain-colonizing Fusarium species on wheat kernels. Since numerous metabolites produced by Fusarium spp. are toxic to insects, we tested the hypothesis that the insect senses and avoids Fusarium-colonized grains. We found that only kernels colonized with F. avenaceum or Beauveria bassiana (an insect-pathogenic fungal control) were avoided by the larvae as expected. Kernels colonized with F. proliferatum, F. poae or F. culmorum attracted T. molitor larvae significantly more than control kernels. The avoidance/preference correlated with larval feeding behaviors and weight gain. Interestingly, larvae that had consumed F. proliferatum- or F. poae-colonized kernels had similar survival rates as control. Larvae fed on F. culmorum-, F. avenaceum- or B. bassiana-colonized kernels had elevated mortality rates. HPLC analyses confirmed the following mycotoxins produced by the fungal strains on the kernels: fumonisins, enniatins and beauvericin by F. proliferatum, enniatins and beauvericin by F. poae, enniatins by F. avenaceum, and deoxynivalenol and zearalenone by F. culmorum. Our results indicate that T. molitor larvae have the ability to sense potential survival threats of kernels colonized with F. avenaceum or B. bassiana, but not with F. culmorum. Volatiles potentially along with gustatory cues produced by these fungi may represent survival threat signals for the larvae resulting in their avoidance. Although F. proliferatum or F. poae produced fumonisins, enniatins and beauvericin during kernel colonization, the larvae were able to use those kernels as diet without exhibiting increased mortality. Consumption of F. avenaceum-colonized kernels, however, increased larval mortality; these kernels had higher enniatin levels than F. proliferatum or F. poae-colonized ones suggesting that T. molitor can tolerate or metabolize those toxins.
doi:10.1371/journal.pone.0100112
PMCID: PMC4059719  PMID: 24932485
2.  Symbiont-Mediated Protection against Fungal Pathogens in Pea Aphids: a Role for Pathogen Specificity? 
Here we show that a bacterial endosymbiont, Regiella insecticola, protects pea aphids (Acyrthosiphon pisum) from the aphid-specific fungal entomopathogen Zoophthora occidentalis but not from the generalist insect fungal pathogen Beauveria bassiana. This finding highlights the complex influence of fungi on the dynamics of this economically important agricultural pest.
doi:10.1128/AEM.03193-12
PMCID: PMC3623210  PMID: 23354709
3.  Exposure to Bacterial Signals Does Not Alter Pea Aphids’ Survival upon a Second Challenge or Investment in Production of Winged Offspring 
PLoS ONE  2013;8(8):e73600.
Pea aphids have an obligate nutritional symbiosis with the bacteria Buchneraaphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes. One possibility is that immune priming with microbial elicitors could stimulate immune protection against subsequent bacterial infections, as has been observed in several other insect systems. To address this hypothesis we challenged aphids with bacterial immune elicitors twenty-four hours prior to live bacterial pathogen infections and then compared their survival rates to aphids that were not pre-exposed to bacterial signals. Using two aphid genotypes, we found no evidence for immune protection conferred by immune priming during infections with either Serratia marcescens or with Escherichia coli. Immune priming was not altered by the presence of facultative, beneficial symbionts in the aphids. In the absence of inducible immune protection, aphids may allocate energy towards other defense traits, including production of offspring with wings that could escape deteriorating conditions. To test this, we monitored the ratio of winged to unwinged offspring produced by adult mothers of a single clone that had been exposed to bacterial immune elicitors, to live E. coli infections or to no challenge. We found no correlation between immune challenge and winged offspring production, suggesting that this mechanism of defense, which functions upon exposure to fungal pathogens, is not central to aphid responses to bacterial infections.
doi:10.1371/journal.pone.0073600
PMCID: PMC3756975  PMID: 24009760
4.  Next Generation Sequencing Based Transcriptome Analysis of Septic-Injury Responsive Genes in the Beetle Tribolium castaneum 
PLoS ONE  2013;8(1):e52004.
Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.
doi:10.1371/journal.pone.0052004
PMCID: PMC3541394  PMID: 23326321
5.  Horizontally transferred fungal carotenoid genes in the two-spotted spider mite Tetranychus urticae 
Biology Letters  2011;8(2):253-257.
Carotenoids are organic pigments commonly synthesized by plants, algae and some micro-organisms. Through absorption of light energy, carotenoids facilitate photosynthesis and provide protection against photo-oxidation. While it was presumed that all carotenoids in animals were sequestered from their diets, aphids were recently shown to harbour genomic copies of functional carotenoid biosynthesis genes that were acquired via horizontal gene transfer from fungi. Our search of available animal transcripts revealed the presence of two related genes in the two-spotted spider mite Tetranychus urticae. Phylogenetic analyses suggest that the T. urticae genes were transferred from fungi into the spider mite genome, probably in a similar manner as recently suggested for aphids. The genes are expressed in both green and red morphs, with red morphs exhibiting higher levels of gene expression. Additionally, there appear to be changes in the expression of these genes during diapause. As carotenoids are associated with diapause induction in these animals, our results add to recent findings highlighting the importance of eukaryotic horizontal gene transfer in the ecology and evolution of higher animals.
doi:10.1098/rsbl.2011.0704
PMCID: PMC3297373  PMID: 21920958
Acyrthosiphon pisum; carotene; lateral gene transfer; mites; pigments
6.  A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella 
BMC Genomics  2011;12:308.
Background
The larvae of the greater wax moth Galleria mellonella are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in Galleria, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing.
Results
We performed a Galleria transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (E ≤ e-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis.
Conclusion
Here, we have developed extensive transcriptomic resources for Galleria. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in Galleria and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.
doi:10.1186/1471-2164-12-308
PMCID: PMC3224240  PMID: 21663692
7.  Galleria mellonella as a Model System for Studying Listeria Pathogenesis▿  
Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L. monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.
doi:10.1128/AEM.01301-09
PMCID: PMC2798647  PMID: 19897755
8.  Immunity and other defenses in pea aphids, Acyrthosiphon pisum 
Genome Biology  2010;11(2):R21.
The genome of the pea aphid Acyrthosiphon pisum lacks genes thought to be crucial in other insects for recognition, signaling and killing of microbes.
Background
Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models.
Results
Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes.
Conclusions
The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection.
doi:10.1186/gb-2010-11-2-r21
PMCID: PMC2872881  PMID: 20178569
9.  Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens 
Journal of Experimental Botany  2009;60(14):4105-4114.
The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.
doi:10.1093/jxb/erp240
PMCID: PMC2755027  PMID: 19734262
Antimicrobial peptides; ascomycete fungi; barley; disease resistance; metchnikowin
10.  MMPs Regulate both Development and Immunity in the Tribolium Model Insect 
PLoS ONE  2009;4(3):e4751.
Background
Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood.
Methodology/Principal Findings
We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19.
Conclusions/Significance
This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs.
doi:10.1371/journal.pone.0004751
PMCID: PMC2649432  PMID: 19270735
11.  Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris 
Background
The planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris have emerged as valuable model organisms in regeneration and stem cell research because of their prominent ability to regenerate a complete organism from any small body fragment. Under natural conditions wounding may result from predator attacks. These injuries open their innermost to a wide array of microbes present in the environment. Therefore, we established the hypothesis that regeneration processes may be linked to or at least accompanied by innate immune responses. In order to screen for septic wounding inducible genes we dissected individuals using a scalpel in the presence of a crude bacterial lipopolysaccharide preparation that is commonly used to elicit innate immune responses in animals and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes.
Results
This analysis revealed the induced expression of 27 genes in immune challenged Schmidtea and 35 genes in immune challenged Hydra. Identified genes from both animals encode proteins that share sequence similarities with potential homologues from other organisms known to be involved in signaling (e.g. calreticulin in Schmidtea and major vault protein in Hydra), stress responses (e.g. Hsp20 in Schmidtea and a PRP19/PSO4 DNA repair protein in Hydra), or to represent potential antimicrobial effectors (e.g. perforin-like protein in Schmidtea and PR-1-like protein and neutrophil cytosolic factor 1 in Hydra). As expected, septic wounding also induces expression of genes in Schmidtea and Hydra potentially involved in tissue remodeling associated with regeneration processes (e.g. matrix metalloproteinase in Schmidtea and a potential von Willebrand factor in Hydra).
Conclusion
We identified numerous immune-inducible genes in Hydra and Schmidtea that show a similar distribution corresponding to their physiological roles, although lineages of both animals split from their common ancestor for more than five hundred millions of years. The present study is the first analysis of immune-inducible genes of these two phylogenetically distant model organisms of regeneration and provide numerous candidate genes that we can use as a starting point for comparative examination of interrelationships between immunity and homeostasis.
doi:10.1186/1742-9994-5-6
PMCID: PMC2386466  PMID: 18439314
12.  Analysis of the immune-inducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly Eristalis tenax 
BMC Genomics  2007;8:326.
Background
The saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Insecta, Diptera) have evolved the unique ability to survive in aquatic habitats with extreme microbial stress such as drains, sewage pools, and farmyard liquid manure storage pits. Therefore, they represent suitable models for the investigation of trade-offs between the benefits resulting from colonization of habitats lacking predators, parasitoids, or competitors and the investment in immunity against microbial stress. In this study, we screened for genes in E. tenax that are induced upon septic injury. Suppression subtractive hybridization was performed to selectively amplify and identify cDNAs that are differentially expressed in response to injected crude bacterial endotoxin (LPS).
Results
Untreated E. tenax maggots exhibit significant antibacterial activity in the hemolymph which strongly increases upon challenge with LPS. In order to identify effector molecules contributing to this microbial defense we constructed a subtractive cDNA library using RNA samples from untreated and LPS injected maggots. Analysis of 288 cDNAs revealed induced expression of 117 cDNAs corresponding to 30 novel gene clusters in E. tenax. Among these immune-inducible transcripts we found homologues of known genes from other Diptera such as Drosophila and Anopheles that mediate pathogen recognition (e.g. peptidoglycan recognition protein) or immune-related signaling (e.g. relish). As predicted, we determined a high diversity of novel putative antimicrobial peptides including one E. tenax defensin.
Conclusion
We identified 30 novel genes of E. tenax that were induced in response to septic injury including novel putative antimicrobial peptides. Further analysis of these immune-related effector molecules from Eristalis may help to elucidate the interdependency of ecological adaptation and molecular evolution of the innate immunity in Diptera.
doi:10.1186/1471-2164-8-326
PMCID: PMC2039750  PMID: 17875201
13.  Analysis of the immune-related transcriptome of a lophotrochozoan model, the marine annelid Platynereis dumerilii 
Frontiers in Zoology  2007;4:18.
Background
The marine annelid Platynereis dumerilii (Polychaeta, Nereididae) has been recognized as a slow-evolving lophotrochozoan that attracts increasing attention as a valuable model for evolutionary and developmental research. Here, we analyzed its immune-related transcriptome. For targeted identification of immune-induced genes we injected bacterial lipopolysaccharide, a commonly used elicitor of innate immune responses, and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes.
Results
Sequence analysis of 288 cDNAs revealed induced expression of numerous genes whose potential homologues from other animals mediate recognition of infection (e.g. complement receptor CD35), signaling (e.g. myc and SOCS), or act as effector molecules like ferritins and the bactericidal permeability-increasing protein. Interestingly, phylogenetic analyses implicate that immune-related genes identified in P. dumerilii are more related to counterparts from Deuterostomia than are those from Ecdysozoa, similarly as recently described for opsin and intron-rich genes.
Conclusion
Obtained results may allow for a better understanding of Platynereis immunity and support the view that P. dumerilii represents a suitable model for analyzing immune responses of Lophotrochozoa.
doi:10.1186/1742-9994-4-18
PMCID: PMC1939704  PMID: 17617895
14.  Microbial Metalloproteinases Mediate Sensing of Invading Pathogens and Activate Innate Immune Responses in the Lepidopteran Model Host Galleria mellonella▿  
Infection and Immunity  2006;75(1):175-183.
Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since G. mellonella was recently established as a valuable pathogenicity model for Cryptococcus neoformans infection, the present results add to our understanding of the mechanisms of immune responses in G. mellonella. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.
doi:10.1128/IAI.01385-06
PMCID: PMC1828416  PMID: 17074843
15.  GcpE Is Involved in the 2-C-Methyl-d-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli 
Journal of Bacteriology  2001;183(8):2411-2416.
In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.
doi:10.1128/JB.183.8.2411-2416.2001
PMCID: PMC95155  PMID: 11274098
16.  Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily 
Molecular and Cellular Biology  1999;19(5):3383-3394.
Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor’s silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.
PMCID: PMC84131  PMID: 10207062

Results 1-16 (16)