Philippe Fournier

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges

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.

Cloning of the LYS5 gene encoding saccharopine dehydrogenase from the yeast Yarrowia lipolytica by target integration

SummaryA Yarrowia lipolytica yeast gene bank has been constructed in E. coli in an integrative plasmid vector containing the homologous LEU2 gene, and used to transform a leu2 lys5 yeast strain. The LYS5 gene encoding saccharopine dehydrogenase (SDH) has been rescued in E. coli from the chromosome of prototrophic transformants, in which the hybrid plasmid had integrated at the leu2 locus. Evidence that the rescued clone contains the LYS5 gene comes from complementation tests, genetic crosses, and SDH assay. Further characterization of the gene has been achieved by deletion mapping and subcloning, and by demonstrating the presence of transcripts hybridizing to this sequence.

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements

The recent assembly of the silkworm Bombyx mori genome with 432 Mb on 28 holocentric chromosomes has become a reference in the genomic analysis of the very diverse Order of Lepidoptera. We sequenced BACs from two major pests, the noctuid moths Helicoverpa armigera and Spodoptera frugiperda, corresponding to 15 regions distributed on 11 B. mori chromosomes, each BAC/region being anchored by known orthologous gene(s) to analyze syntenic relationships and genome rearrangements among the three species. Nearly 300 genes and numerous transposable elements were identified, with long interspersed nuclear elements and terminal inverted repeats the most abundant transposable element classes. There was a high degree of synteny conservation between B. mori and the two noctuid species. Conserved syntenic blocks of identified genes were very small, however, approximately 1.3 genes per block between B. mori and the two noctuid species and 2.0 genes per block between S. frugiperda and H. armigera. This corresponds to approximately two chromosome breaks per Mb DNA per My. This is a much higher evolution rate than among species of the Drosophila genus and may be related to the holocentric nature of the lepidopteran genomes. We report a large cluster of eight members of the aminopeptidase N gene family that we estimate to have been present since the Jurassic. In contrast, several clusters of cytochrome P450 genes showed multiple lineage-specific duplication events, in particular in the lepidopteran CYP9A subfamily. Our study highlights the value of the silkworm genome as a reference in lepidopteran comparative genomics.

High frequency of yeast transformation by plasmids carrying part of entire 2-μm yeast plasmid

Abstract By using two chimeric plasmids containing yeast ura3 gene and 2-μm yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-μm DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-μm—ura3 sequence can be considered as a “transposable” block. In contrast, the second one bears the entire 2-μm plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-μm DNA of the recipient strain. The specific activity of orotidine 5′-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.

SPODOBASE : an EST database for the lepidopteran crop pest Spodoptera

BackgroundThe 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.DescriptionThe 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.ConclusionThe 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

Characterization of the cDNA encoding the 90 kDa heat-shock protein in the Lepidoptera Bombyx mori and Spodoptera frugiperda

This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).

AN ORIGIN OF REPLICATION AND A CENTROMERE ARE BOTH NEEDED TO ESTABLISH A REPLICATIVE PLASMID IN THE YEAST YARROWIA LIPOLYTICA

Two DNA fragments displaying ARS activity on plasmids in the yeast Yarrowia lipolytica have previously been cloned and shown to harbor centromeric sequences (P. Fournier, A. Abbas, M. Chasles, B. Kudla, D. M. Ogrydziak, D. Yaver, J.-W. Xuan, A. Peito, A.-M. Ribet, C. Feynerol, F. He, and C. Gaillardin, Proc. Natl. Acad. Sci. USA 90:4912-4916, 1993; and P. Fournier, L. Guyaneux, M. Chasles, and C. Gaillardin, Yeast 7:25-36, 1991). We have used the integration properties of centromeric sequences to show that all Y. lipolytica ARS elements so far isolated are composed of both a replication origin and a centromere. The sequence and the distance between the origin and centromere do not seem to play a critical role, and many origins can function in association with one given centromere. A centromeric plasmid can therefore be used to clone putative chromosomal origins coming from several genomic locations, which confer the replicative property on the plasmid. The DNA sequences responsible for initiation in plasmids are short (several hundred base pairs) stretches which map close to or at replication initiation sites in the chromosome. Their chromosomal deletion abolishes initiation, but changing their chromosomal environment does not.

Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels

BackgroundGenomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV.ResultsResults show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76%) showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses).ConclusionThis analysis of the host-polydnavirus interactions by a microarray approach indicates that the presence of HdIV induces, directly or indirectly, variations in transcript levels of specific host genes, changes that could be responsible in part for the alterations observed in the parasitized host physiology. Development of such global approaches will allow a better understanding of the strategies employed by parasites to manipulate their host physiology, and will permit the identification of potential targets of the immunosuppressive polydnaviruses.

Characterization and transcriptional profiles of three Spodoptera frugiperda genes encoding cysteine-rich peptides. A new class of defensin-like genes from lepidopteran insects?

The present work describes sequence and transcription of three Spodoptera frugiperda genes encoding 6-cysteine-rich peptides. Sequence alignments indicate that the predicted peptides belong to the insect defensin family, although phylogenetic analyses suggest they form a cluster distinct from that of other neopteran insect defensins. The three genes were identified in a non-immune-challenged Sf9 cells cDNA (DNA complementary to RNA) library (Landais et al., Bioinformatics, in press) and were named spodoptericin, Sf-gallerimycin and Sf-cobatoxin. Spodoptericin is a novel defensin-like gene that appears to be weakly up-regulated following injection of bacteria and fungi. Interestingly, no sequence motif clearly homologous to cis regulatory element involved in the regulation of antimicrobial genes was found. An homologue of the spodoptericin gene was identified in the SilkBase Bombyx mori cDNA library. Sf-gallerimycin is related to the Galleria mellonella gallerimycin gene and is induced after immune challenge by injection of bacteria in the larval fat body as well as in hemocytes. In silico analysis of the sequence upstream from the cDNA reveals the presence of at least one motif homologous to a nuclear factor kappaB (NF-kappaB) binding site. Finally, Sf-cobatoxin is related to the G. mellonella cobatoxin-like gene. Despite high levels of constitutive expression compared to the two previous genes, transcription of Sf-cobatoxin is increased after immune, in particular, bacterial challenge. We therefore confirm that these three genes encode potential candidate molecules involved in S. frugiperda innate humoral response.

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.