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Characterization of Hovi-mEH1, a microsomal epoxide hydrolase from the glassy-winged sharpshooter Homalodisca vitripennis.

ABSTRACT: Epoxide hydrolase (EH) is an enzyme in the ?/?-hydrolase fold superfamily that uses a water molecule to transform an epoxide to its corresponding diol. In insects, EHs metabolize among other things critical developmental hormones called juvenile hormones (JHs). EHs also play roles in the detoxification of toxic compounds that are found in the insect's diet or environment. In this study, a full-length cDNA encoding an epoxide hydrolase, Hovi-mEH1, was obtained from the xylem-feeding insect Homalodisca vitripennis. H. vitripennis, commonly known as the glassy-winged sharpshooter, is an economically important vector of plant pathogenic bacteria such as Xylella fastidiosa. Hovi-mEH1 hydrolyzed the general EH substrates cis-stilbene oxide and trans-diphenylpropene oxide with specific activities of 47.5 ± 6.2 and 1.3 ± 0.5 nmol of diol formed min?¹ mg?¹, respectively. Hovi-mEH1 metabolized JH III with a Vmax of 29.3 ± 1.6 nmol min?¹ mg?¹, kcat of 0.03 s?¹, and KM of 13.8 ± 2.0 ?M. These Vmax and kcat values are similar to those of known JH metabolizing EHs from lepidopteran and coleopteran insects. Hovi-mEH1 showed 99.1% identity to one of three predicted EH-encoding sequences that were identified in the transcriptome of H. vitripennis. Of these three sequences only Hovi-mEH1 clustered with known JH metabolizing EHs. On the basis of biochemical, phylogenetic, and transcriptome analyses, we hypothesize that Hovi-mEH1 is a biologically relevant JH-metabolizing enzyme in H. vitripennis.

SUBMITTER: Kamita SG

PROVIDER: S-EPMC3846607 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Characterization of Hovi-mEH1, a microsomal epoxide hydrolase from the glassy-winged sharpshooter Homalodisca vitripennis.

Kamita Shizuo G SG Oshita Grant H GH Wang Peng P Morisseau Christophe C Hammock Bruce D BD Nandety Raja Sekhar RS Falk Bryce W BW

Archives of insect biochemistry and physiology 20130523 4

Epoxide hydrolase (EH) is an enzyme in the α/β-hydrolase fold superfamily that uses a water molecule to transform an epoxide to its corresponding diol. In insects, EHs metabolize among other things critical developmental hormones called juvenile hormones (JHs). EHs also play roles in the detoxification of toxic compounds that are found in the insect's diet or environment. In this study, a full-length cDNA encoding an epoxide hydrolase, Hovi-mEH1, was obtained from the xylem-feeding insect Homalo ...[more]

PMID: 23704009

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Project description:BackgroundThe glassy-winged sharpshooter Homalodisca vitripennis (Hemiptera: Cicadellidae), is a xylem-feeding leafhopper and important vector of the bacterium Xylella fastidiosa; the causal agent of Pierce's disease of grapevines. The functional complexity of the transcriptome of H. vitripennis has not been elucidated thus far. It is a necessary blueprint for an understanding of the development of H. vitripennis and for designing efficient biorational control strategies including those based on RNA interference.ResultsHere we elucidate and explore the transcriptome of adult H. vitripennis using high-throughput paired end deep sequencing and de novo assembly. A total of 32,803,656 paired-end reads were obtained with an average transcript length of 624 nucleotides. We assembled 32.9 Mb of the transcriptome of H. vitripennis that spanned across 47,265 loci and 52,708 transcripts. Comparison of our non-redundant database showed that 45% of the deduced proteins of H. vitripennis exhibit identity (e-value ≤1(-5)) with known proteins. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript isoform. In order to gain insight into the molecular basis of key regulatory genes of H. vitripennis, we characterized predicted proteins involved in the metabolism of juvenile hormone, and biogenesis of small RNAs (Dicer and Piwi sequences) from the transcriptomic sequences. Analysis of transposable element sequences of H. vitripennis indicated that the genome is less expanded in comparison to many other insects with approximately 1% of the transcriptome carrying transposable elements.ConclusionsOur data significantly enhance the molecular resources available for future study and control of this economically important hemipteran. This transcriptional information not only provides a more nuanced understanding of the underlying biological and physiological mechanisms that govern H. vitripennis, but may also lead to the identification of novel targets for biorationally designed control strategies.

Project description:A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter, Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5(th) instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of beta-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly alpha-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.

Project description:BackgroundHomalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure.ResultsWe identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here.ConclusionsIn this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

Dataset Information

Characterization of Hovi-mEH1, a microsomal epoxide hydrolase from the glassy-winged sharpshooter Homalodisca vitripennis.

Publications

Characterization of Hovi-mEH1, a microsomal epoxide hydrolase from the glassy-winged sharpshooter Homalodisca vitripennis.

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