RESUMO
BACKGROUND: Eukaryotic genomes are packaged by Histone proteins in a structure called chromatin. There are different chromatin types. Euchromatin is typically associated with decondensed, transcriptionally active regions and heterochromatin to more condensed regions of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved biochemical marker of heterochromatin. In many organisms, heterochromatin is usually localized at telomeric as well as pericentromeric regions but can also be found at interstitial chromosomal loci. This distribution may vary in different species depending on their general chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera: Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no observable pericentromeric compartment. To identify the localization of heterochromatin in such species we performed ChIP-Seq experiments and analyzed the distribution of the heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4th instar larvae (L4) in relation to RNA-Seq data. RESULTS: In both samples we measured an enrichment of H3K9me2 at the (sub) telomeres, rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions. We also observed that density of H3K9me2 is positively correlated with transposable elements and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not correlated with transcriptional repression. CONCLUSION: This is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for H3K9me2. Compared to model organisms, this mark is found in expected chromosomal compartments such as rDNA and telomeres. However, it is also localized at numerous dispersed regions, instead of the well described large pericentromeric domains, indicating that H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera. (242 words).
Assuntos
Heterocromatina , Histonas , Animais , Cromatina , Elementos de DNA Transponíveis , Heterocromatina/genética , Histonas/metabolismo , Spodoptera/genética , Spodoptera/metabolismoRESUMO
BACKGROUND: The degree to which adaptation to same environment is determined by similar molecular mechanisms, is a topic of broad interest in evolutionary biology, as an indicator of evolutionary predictability. We wished to address if adaptation to the same host plant in phytophagous insects involved related gene expression patterns. We compared sRNA-Seq and RNA-Seq data between two pairs of taxa of Ostrinia and Spodoptera frugiperda sharing maize as host-plant. For the latter, we had previously carried out a reciprocal transplant experiment by feeding of the larvae of the Corn strain (Sf-C) and the Rice strain (Sf-R) on corn versus rice and characterized the mRNA and miRNA responses. RESULTS: First, we predicted the genes encoding miRNA in Ostrinia nubilalis (On) and O. scapulalis (Os). Respectively 67 and 65 known miRNA genes, as well as 196 and 190 novel ones were predicted with Os genome using sncRNAs extracted from whole larvae feeding on corn or mugwort. In On, a read counts analysis showed that 37 (55.22%) known miRNAs and 19 (9.84%) novel miRNAs were differentially expressed (DE) on mugwort compared to corn (in Os, 25 known miRs (38.46%) and 8 novel ones (4.34%)). Between species on corn, 8 (12.5%) known miRNAs and 8 (6.83%) novel ones were DE while only one novel miRNA showed expression variation between species on mugwort. Gene target prediction led to the identification of 2953 unique target genes in On and 2719 in Os, among which 11.6% (344) were DE when comparing species on corn. 1.8% (54) of On miR targets showed expression variation upon a change of host-plant. We found molecular changes matching convergent phenotype, i.e., a set of nine miRNAs that are regulated either according to the host-plant both in On and Sf-C or between them on the same plant, corn. Among DE miR target genes between taxa, 13.7% shared exactly the same annotation between the two pairs of taxa and had function related to insect host-plant interaction. CONCLUSION: There is some similarity in underlying genetic mechanisms of convergent evolution of two distant Lepidopteran species having adopted corn in their host range, highlighting possible adaptation genes.
Assuntos
MicroRNAs , Mariposas , Agricultura , Animais , Perfilação da Expressão Gênica , MicroRNAs/genética , Mariposas/genética , Transcriptoma , Zea mays/genéticaRESUMO
The noctuid genus Spodoptera currently consists of 31 species with varied host plant breadths, ranging from monophagous and oligophagous non-pest species to polyphagous pests of economic importance. Several of these pest species have become major invaders, colonizing multiple continents outside their native range. Such is the case of the infamous fall armyworm, Spodoptera frugiperda (J.E. Smith), which includes two recognized host strains that have not been treated as separate species. Following its accidental introduction to Africa in 2016, it quickly spread through Africa and Asia to Australia. Given that half the described Spodoptera species cause major crop losses, comparative genomics studies of several Spodoptera species have highlighted major adaptive changes in genetic architecture, possibly relating to their pest status. Several recent population genomics studies conducted on two species enable a more refined understanding of their population structures, migration patterns and invasion processes. Despite growing interest in the genus, the taxonomic status of several Spodoptera species remains unstable and evolutionary studies suffer from the absence of a robust and comprehensive dated phylogenetic framework. We generated mitogenomic data for 14 Spodoptera taxa, which are combined with data from 15 noctuoid outgroups to generate a resolved mitogenomic backbone phylogeny using both concatenation and multi-species coalescent approaches. We combine this backbone with additional mitochondrial and nuclear data to improve our understanding of the evolutionary history of the genus. We also carry out comprehensive dating analyses, which implement three distinct calibration strategies based on either primary or secondary fossil calibrations. Our results provide an updated phylogenetic framework for 28 Spodoptera species, identifying two well-supported ecologically diverse clades that are recovered for the first time. Well-studied larvae in each of these clades are characterized by differences in mandibular shape, with one clade's being more specialized on silica-rich C4 grasses. Interestingly, the inferred timeframe for the genus suggests an earlier origin than previously thought for the genus: about 17-18 million years ago.
Assuntos
Evolução Molecular , Filogenia , Spodoptera/classificação , Spodoptera/genética , Animais , Interações Hospedeiro-Parasita , FilogeografiaRESUMO
BACKGROUND: A change in the environment may impair development or survival of living organisms leading them to adapt to the change. The resulting adaptation trait may reverse, or become fixed in the population leading to evolution of species. Deciphering the molecular basis of adaptive traits can thus give evolutionary clues. In phytophagous insects, a change in host-plant range can lead to emergence of new species. Among them, Spodoptera frugiperda is a major agricultural lepidopteran pest consisting of two host-plant strains having diverged 3 MA, based on mitochondrial markers. In this paper, we address the role of microRNAs, important gene expression regulators, in response to host-plant change and in adaptive evolution. RESULTS: Using small RNA sequencing, we characterized miRNA repertoires of the corn (C) and rice (R) strains of S. frugiperda, expressed during larval development on two different host-plants, corn and rice, in the frame of reciprocal transplant experiments. We provide evidence for 76 and 68 known miRNAs in C and R strains and 139 and 171 novel miRNAs. Based on read counts analysis, 34 of the microRNAs were differentially expressed in the C strain larvae fed on rice as compared to the C strain larvae fed on corn. Twenty one were differentially expressed on rice compared to corn in R strain. Nine were differentially expressed in the R strain compared to C strain when reared on corn. A similar ratio of microRNAs was differentially expressed between strains on rice. We could validate experimentally by QPCR, variation in expression of the most differentially expressed candidates. We used bioinformatics methods to determine the target mRNAs of known microRNAs. Comparison with the mRNA expression profile during similar reciprocal transplant experiment revealed potential mRNA targets of these host-plant regulated miRNAs. CONCLUSIONS: In the current study, we performed the first systematic analysis of miRNAs in Lepidopteran pests feeding on host-plants. We identified a set of the differentially expressed miRNAs that respond to the plant diet, or differ constitutively between the two host plant strains. Among the latter, the ones that are also deregulated in response to host-plant are molecular candidates underlying a complex adaptive trait.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Insetos/genética , MicroRNAs/genética , Oryza/parasitologia , Spodoptera/genética , Zea mays/parasitologia , Animais , Biologia Computacional , Comportamento Alimentar , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Hospedeiro , Larva , Spodoptera/classificaçãoRESUMO
The moth Spodoptera frugiperda is a well-known pest of crops throughout the Americas, which consists of two strains adapted to different host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. Though morphologically indistinguishable, they exhibit differences in their mating behavior, pheromone compositions, and show development variability according to the host-plant. Though the latter suggest that both strains are different species, this issue is still highly controversial because hybrids naturally occur in the wild, not to mention the discrepancies among published results concerning mating success between the two strains. In order to clarify the status of the two host-plant strains of S. frugiperda, we analyze features that possibly reflect the level of post-zygotic isolation: (1) first generation (F1) hybrid lethality and sterility; (2) patterns of meiotic segregation of hybrids in reciprocal second generation (F2), as compared to the meiosis of the two parental strains. We found a significant reduction of mating success in F1 in one direction of the cross and a high level of microsatellite markers showing transmission ratio distortion in the F2 progeny. Our results support the existence of post-zygotic reproductive isolation between the two laboratory strains and are in accordance with the marked level of genetic differentiation that was recovered between individuals of the two strains collected from the field. Altogether these results provide additional evidence in favor of a sibling species status for the two strains.
Assuntos
Cruzamentos Genéticos , Especificidade de Hospedeiro , Spodoptera/classificação , Animais , Feminino , Fertilidade/genética , Marcadores Genéticos , Técnicas de Genotipagem , Hibridização Genética , Masculino , Repetições de Microssatélites , Oryza , Spodoptera/genética , Zea maysRESUMO
BACKGROUND: Spodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides. RESULTS: In this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family--ribosomal proteins--as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied. CONCLUSION: We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae.
Assuntos
Perfilação da Expressão Gênica/normas , Spodoptera/genética , Transcriptoma , Animais , Genes de Insetos , Imunidade Inata/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Anotação de Sequência Molecular , Padrões de Referência , Olfato/genética , Spodoptera/metabolismoRESUMO
BACKGROUND: Divergent selection on host-plants is one of the main evolutionary forces driving ecological speciation in phytophagous insects. The ecological speciation might be challenging in the presence of gene flow and assortative mating because the direction of divergence is not necessarily the same between ecological selection (through host-plant adaptation) and assortative mating. The fall armyworm (FAW), a major lepidopteran pest species, is composed of two sympatric strains, corn and rice strains, named after two of their preferred host-plants. These two strains have been hypothesized to undergo incipient speciation, based on (i) several lines of evidence encompassing both pre- and post-zygotic reproductive isolation, and (ii) the presence of a substantial level of genetic differentiation. Even though the status of these two strains has been established a long time ago, it is still yet to be found whether these two strains indeed exhibit a marked level of genetic differentiation from a large number of genomic loci. Here, we analyzed whole genome sequences from 56 FAW individuals either collected from pasture grasses (a part of the favored host range of the rice strain) or corn to assess the role of host-plant adaptation in incipient speciation. RESULTS: Principal component analysis of whole genome data shows that the pattern of divergence in the fall armyworm is predominantly explained by the genetic differentiation associated with host-plants. The level of genetic differentiation between corn and rice strains is particularly marked in the Z chromosome. We identified one autosomal locus and two Z chromosome loci targeted by selective sweeps specific to rice strain and corn strain, respectively. The autosomal locus has both increased DXY and FST while the Z chromosome loci had decreased DXY and increased FST. CONCLUSION: These results show that the FAW population structure is dominated by the genetic differentiation between corn and rice strains. This differentiation involves divergent selection targeting at least three loci, which include a locus potentially causing reproductive isolation. Taken together, these results suggest the evolutionary scenario that host-plant speciation is a driver of incipient speciation in the fall armyworm.
Assuntos
Oryza , Zea mays , Humanos , Animais , Spodoptera/genética , Zea mays/genética , Isolamento Reprodutivo , Fluxo Gênico/genética , Oryza/genéticaRESUMO
The fall armyworm (FAW; Spodoptera frugiperda) is one of the major agricultural pest insects. FAW is native to the Americas, and its invasion was first reported in West Africa in 2016. Then it quickly spread through Africa, Asia, and Oceania, becoming one of the main threats to corn production. We analyzed whole genome sequences of 177 FAW individuals from 12 locations on four continents to infer evolutionary processes of invasion. Principal component analysis from the TPI gene and whole genome sequences shows that invasive FAW populations originated from the corn strain. Ancestry coefficient and phylogenetic analyses from the nuclear genome indicate that invasive populations are derived from a single ancestry, distinct from native populations, while the mitochondrial phylogenetic tree supports the hypothesis of multiple introductions. Adaptive evolution specific to invasive populations was observed in detoxification, chemosensory, and digestion genes. We concluded that extant invasive FAW populations originated from the corn strain with potential contributions of adaptive evolution.
Assuntos
Spodoptera , Humanos , Animais , Spodoptera/genética , Filogenia , Ásia , África , África OcidentalRESUMO
Understanding the genetic basis of insecticide resistance is a key topic in agricultural ecology. The adaptive evolution of multi-copy detoxification genes has been interpreted as a cause of insecticide resistance, yet the same pattern can also be generated by the adaptation to host-plant defense toxins. In this study, we tested in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), if adaptation by copy number variation caused insecticide resistance in two geographically distinct populations with different levels of resistance and the two host-plant strains. We observed a significant allelic differentiation of genomic copy number variations between the two geographic populations, but not between host-plant strains. A locus with positively selected copy number variation included a CYP gene cluster. Toxicological tests supported a central role for CYP enzymes in deltamethrin resistance. Our results indicate that copy number variation of detoxification genes might be responsible for insecticide resistance in fall armyworm and that evolutionary forces causing insecticide resistance could be independent of host-plant adaptation.
Assuntos
Variações do Número de Cópias de DNA/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Spodoptera , Animais , Sistema Enzimático do Citocromo P-450/genética , Feminino , Genoma de Inseto/genética , Nitrilas/farmacologia , Piretrinas/farmacologia , Spodoptera/efeitos dos fármacos , Spodoptera/genéticaRESUMO
Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.
Assuntos
Adaptação Fisiológica/genética , Genoma de Inseto , Herbivoria , Spodoptera/genética , Animais , Produtos Agrícolas , Larva/genética , Especificidade da EspécieRESUMO
BACKGROUND: The Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations. DESCRIPTION: The ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary. CONCLUSION: The SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL: http://bioweb.ensam.inra.fr/spodobase.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Spodoptera/genética , Animais , Análise por Conglomerados , Mapeamento de Sequências Contíguas , DNA Complementar/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Software , Distribuição TecidualRESUMO
Two genomic tools for the study of Lepidoptera and the holocentric structure of their chromosomes are presented in this paper. A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA partially digested with HindIII from eggs of Spodoptera frugiperda. The library contains a total of 36,864 clones with an average insert size of 125 kb, which corresponds to approximately 11.5 genome equivalents. Hybridization screening of the library was performed with eight single-copy genes, giving an average hit of 10 clones per marker gene. Colinearity between the genome and BACs was demonstrated at the triose phosphate isomerase (tpi) locus. Probing of the library with a PCR fragment internal to the 18S ribosomal gene allowed an estimation of the rDNA locus size close to 115 repeats per haploid genome. A new vector (pBAC3.6eGFP) for transient transfection into S. frugiperda cell lines has been constructed. It is based on the BAC vector, pBAC3.6e, in which a gene encoding GFP was inserted under the control of the densovirus P9 promoter. This vector has the advantage to accommodate large genomic inserts and to be transfected in a large lepidopteran host range. It was used to construct a second BAC library from Sf9 cell nuclear DNA in order to allow a comparison between somatic and cell line genome organization.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Vetores Genéticos/genética , Biblioteca Genômica , Genômica/métodos , Proteínas Luminescentes/genética , Spodoptera/genética , Animais , Linhagem Celular , Cromossomos/genética , Clonagem Molecular , DNA/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Densovirus/genética , Drosophila/genética , Genes de Insetos/genética , Proteínas de Fluorescência Verde , Hibridização In Situ , Lepidópteros/genética , Óvulo , Reação em Cadeia da Polimerase , Spodoptera/citologia , TransfecçãoRESUMO
The proteins of the X-tox family have imperfectly conserved tandem repeats of several defensin-like motifs known as cysteine-stabilized αß (CS-αß) motifs. These immune-related proteins are inducible and expressed principally in hemocytes, but they have lost the antimicrobial properties of the ancestral defensins from which they evolved. We compared x-tox gene structure and expression in three lepidopteran species (Spodoptera frugiperda, Helicoverpa armigera and Bombyx mori). Synteny and phylogenetic analyses showed that the x-tox exons encoding CS-αß motifs were phylogenetically closely related to defensin genes mapping to chromosomal positions close to the x-tox genes. We were able to define two groups of paralogous x-tox exons (three in Noctuids) that each followed the expected species tree. These results suggest that the ancestor of the three species already possessed an x-tox gene with at least two proto-domains, and an additional duplication/fusion should have occurred in the ancestor of the two noctuid species. An expansion of the number of exons subsequently occurred in each lineage. Alternatively, the proto x-tox gene possessed more copy and each group of x-tox domains might undergo concerted evolution through gene conversion. Accelerated protein evolution was detected in x-tox domains when compared to related defensins, concomitantly to multiplication of exons and/or the possible activation of concerted evolution. The x-tox genes of the three species have similar structural organizations, with repeat motifs composed of CS-αß-encoding exons flanked by introns in phase 1. Diverse mechanisms underlie this organization: (i) the acquisition of new repeat motifs, (ii) the duplication of preexisting repeat motifs and (iii) the duplication of modules. A comparison of gDNA and cDNA structures showed that alternative splicing results in the production of multiple X-tox protein isoforms from the x-tox genes. Differences in the number and sequence of CS-αß motifs in these isoforms were found between species, but also between individuals of the same species. Thus, our analysis of the genetic organization and expression of x-tox genes in three lepidopteran species suggests a rapid evolution of the organization of these genes.
Assuntos
Processamento Alternativo , Bombyx/genética , Evolução Molecular , Proteínas de Insetos/genética , Spodoptera/genética , Animais , Sequência de Bases , Bombyx/imunologia , Defensinas/genética , Componentes do Gene , Expressão Gênica , Genoma de Inseto , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Isoformas de Proteínas , Spodoptera/imunologia , SinteniaRESUMO
The discovery of an homolog of the human centromeric protein B, CENP-B, in an EST database of the holocentric insect species Spodoptera frugiperda prompted us to further characterize that gene because i) CENP-B has not been described in invertebrates yet ii) it should be a milestone in the molecular characterization of the holocentric centromere of Lepidoptera. Like its human counterpart, the Sf CENP-B protein is related to the transposase of the pogo transposable element (TE) of D. melanogaster. In this paper, we show evidences that the lepidopteran cenpB gene has evolved from domestication of a transposase. Furthermore, the Sf CENP-B nuclear location and its ability to bind to a retrotransposon derived sequence in vivo argue in favor of a functional homology to CENP-B proteins.
Assuntos
Proteína B de Centrômero/genética , Genoma de Inseto , Proteínas de Insetos/genética , Spodoptera/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Centrômero/genética , Proteína B de Centrômero/metabolismo , Imunoprecipitação da Cromatina , Clonagem Molecular , Códon , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retroelementos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transposases/genética , Transposases/metabolismoRESUMO
Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , RNA Interferente Pequeno/genética , Spodoptera/embriologia , Spodoptera/crescimento & desenvolvimento , Animais , Sequência de Bases , Cromatina/metabolismo , Análise por Conglomerados , Biologia Computacional/métodos , Elementos de DNA Transponíveis , Drosophila melanogaster , Genoma , Heterocromatina/química , Histonas/química , Dados de Sequência Molecular , Mutação , Complexo de Inativação Induzido por RNA/genética , Análise de Sequência de DNA , Spodoptera/metabolismoRESUMO
The invertebrate parvovirus Junonia coenia densovirus (JcDNV) shares similarities with terminal hairpins and nonstructural (NS) protein activities of adeno-associated virus (AAV) despite their evolutionary divergence (B. Dumas, M. Jourdan, A. M. Pascaud, and M. Bergoin, Virology, 191:202-222, 1992, and C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol. 76:338-345, 2002). We demonstrate here that persistent transgene expression in insect cells results from stable integration of transfected JcDNV-derived vectors into the host genome. To assess the integrative properties of JcDNV vectors, the green fluorescent protein (GFP) gfp marker gene was fused in frame into the major open reading frame (ORF1) of the viral sequence under the control of the P9 capsid protein promoter. In addition, the influence of the nonstructural proteins on the posttransfection maintenance of the vectors was examined by interruption of one or all three NS ORFs. Following transfection of Sf9 cells with each of the JcDNV constructs, clones showing persistent GFP expression were isolated. Structural analyses revealed that the majority of the JcDNV plasmid sequence was integrated into the genome of the fluorescent clones. Integration was observed whether or not NS proteins were expressed. However, the presence of NS genes in the constructs greatly influenced the number of integrated copies and their distribution in the host genome. Disruption of NS genes expression resulted in integration of head-to-tail concatemers at multiple sites within the genome. Further analyses demonstrated that the cis JcDNV 5' inverted terminal repeat region was the primary site of recombination. Sequence analyses of integration junctions showed rearrangements of both flanking and internal sequences for most integrations. These findings demonstrate that JcDNV vectors integrate into insect cells in a manner similar to AAV plasmids in mammalian cells.