title: De novo transcriptome assembly and analysis to identify potential gene targets for RNAi-mediated control of the tomato leafminer (Tuta absoluta).
abstract: BACKGROUND
  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 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.
  
  
  RESULTS
  We 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 1(st) instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes.
  
  
  CONCLUSIONS
  The 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 1(st) 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.
authors: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil. camargoroberto22@gmail.com.|Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ), Universidade de São Paulo, Av. Pádua Dias, 11, CP 09, Piracicaba, SP, 13418-900, Brazil. camargoroberto22@gmail.com.
authors: Roberto de A
authors: Rde A
authors: Camargo
authors: Department of Pediatrics, Cellular and Molecular Medicine, School of Medicine, University of California San Diego, Torrey Pines Scenic Dr, La Jolla, CA, 92093-0695, USA. roberto.herai@pucpr.br.|Graduate Program in Health Science, School of Medicine, Pontifícia Universidade Católica do Paraná, R. Imaculada Conceição, 1155, Prado Velho, Curitiba, PR, 80215-901, Brazil. roberto.herai@pucpr.br.
authors: Roberto H
authors: RH
authors: Herai
authors: Instituto de Biologia, Departamento de Histologia e Embriologia, Universidade Estadual de Campinas, R. Charles Darwin, CP 6109, Campinas, SP, 13083-863, Brazil. luana_ns16@yahoo.com.br.
authors: Luana N
authors: LN
authors: Santos
authors: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil. flaviammbento@hotmail.com.|Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ), Universidade de São Paulo, Av. Pádua Dias, 11, CP 09, Piracicaba, SP, 13418-900, Brazil. flaviammbento@hotmail.com.
authors: Flavia M M
authors: FM
authors: Bento
authors: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil. limajoni@googlemail.com.
authors: Joni E
authors: JE
authors: Lima
authors: Instituto de Biologia, Departamento de Histologia e Embriologia, Universidade Estadual de Campinas, R. Charles Darwin, CP 6109, Campinas, SP, 13083-863, Brazil. hmsouza@unicamp.br.
authors: Henrique
authors: H
authors: Marques-Souza
authors: Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, CP 96, Piracicaba, SP, 13400-970, Brazil. figueira@cena.usp.br.
authors: Antonio
authors: A
authors: Figueira
mesh_terms: D000818:Animals; D001482:Base Composition; D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015723:Gene Library; D020868:Gene Silencing; D017344:Genes, Insect; D059014:High-Throughput Nucleotide Sequencing; D006728:Hormones / Q000096:biosynthesis; D007300:Insect Control / Q000379:methods; D018551:Solanum lycopersicum / Q000469:parasitology; D058977:Molecular Sequence Annotation; D009036:Moths / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D006728:Hormones; D012333:RNA, Messenger