Abstract
BackgroundProviding double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The tomato leafminer (Tuta absoluta Meyrick) is a major Solanum lycopersicum (tomato) pest that causes significant yield losses and has recently invaded Europe, from where it is spreading at an alarming rate. To explore RNA interference (RNAi) against T. absoluta, sequence information on potential target genes is necessary, but only a few sequences are available in public databases.ResultsWe sequenced six libraries from RNA samples from eggs, adults, and larvae at four stages, obtaining an overall total of around 245 million reads. The assembled T. absoluta transcriptome contained 93,477 contigs with an average size of 1,574 bp, 59.8 % of which presented positive Blast hits, with 19,995 (21.4 %) annotated by gene ontology. From the transcriptome, most of the core genes of the RNAi mechanism of Lepidoptera were identified indicating the potential suitability of T. absoluta for gene silencing. No contigs displayed significant similarity with a RNA-dependent RNA Polymerase. Genes from the juvenile hormone and ecdysteroid biosynthetic pathways were identified, representing potential target genes for systemic silencing. Comparisons of transcript profiles among stages revealed 1,577 genes differentially expressed at earlier larval stages, from which potential gene targets were identified. Five of these genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, which were fed to 1st instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes.ConclusionsThe transcriptome we generated represents a valuable genomic resource for screening potential gene targets that affect the development or survival of T. absoluta larvae. Five novel genes that showed greater expression at the 1st larval stage were demonstrated to be effective potential RNAi targets by reducing larval weight and can be considered good candidates for use in RNAi-mediated crop protection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1841-5) contains supplementary material, which is available to authorized users.
Highlights
Providing double-stranded RNA to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs
Since the discovery that providing double-stranded RNA to a wide variety of organisms, including insects, can induce RNA interference (RNAi), this method has become a common tool for functional genomic studies, in non-model systems [1, 2]
Comparisons of transcript profiles among stages revealed differentially expressed transcripts (DET) at earlier larval instar stages, which allowed identification of potential gene targets. Five such genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, fed to 1st instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting knockdown of the genes
Summary
Providing double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The demonstration that transgenic plants modified to express insect dsRNA can successfully control target pests [5, 6] raised the possibility of developing RNAi-mediated crop protection [3, 4, 7]. Transgenic plants expressing dsRNA matching specific insect target genes have been demonstrated to control Lepidoptera, Coleoptera and Hemiptera agricultural pests [4], including Helicoverpa armigera in cotton [8] and tobacco [9, 10], Diabrotica virgifera virgifera in maize [5], Nilaparvata lugens in rice [11], Myzus persicae in Nicotiana benthamiana and Arabidopsis thaliana [12], and Sitobion avenae in wheat [13]
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