Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target

Abstract

BackgroundInsect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening.ResultsWe use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.ConclusionsOur results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1880-y) contains supplementary material, which is available to authorized users.

RNA interference: a promising biopesticide strategy against the African Sweetpotato Weevil Cylas brunneus

“…One of the most lethal dsRNAs in our microinjection assays was the one targeting prosα2 , encoding the proteasome alpha type-2 subunit. Ulrich et al 31. concluded in a recent publication that the proteasome could be a prime target for RNAi-based pest control in insects.…”

Comparison of RNAi Sequences in Insect-Resistant Plants to Expressed Sequences of a Beneficial Lady Beetle: A Closer Look at Off-Target Considerations

“…The aa identity was 55/80 or 69% and the nt identity was 188/254 or 74%. The nt alignment did not result in any continuous nt identities of >17 nt, the expected length for predicting possible off-target effects [13,18]. …”

“…The spreadsheet generated was sorted and searched to identify sequences that were potentially homologous or orthologous to those evaluated for RNAi pest control sequences targeting D. v. virgifera [12] and L. decemlineata [7], insects in the same order as C. maculata , Coleoptera, and likely to be present in the same North American agroecosystems, maize and potato. Similarly, sequences designated as prime RNAi targets [13] were compared with the paired C. maculata transcriptomes after matching the T. castaneum sequences to GenBank reference mRNA/cDNA sequences. Candidate C. maculata sequences from each assembly were aligned to one another to verify their identity using the NCBI BLAST comparison of two nucleotides setting.…”

“…Searches of the transcriptomes identified sequences as the most likely to be orthologous to the sequences described in RNAi plant incorporated products. Additionally, eleven proposed prime RNAi pest management targets [13] were used to search for similar sequences in the transcriptomes. The identified C. maculata sequences were aligned with sequences from corn rootworms and CPB as appropriate, and in the case of targets, regions of similarity were compared with the genetic model organism for beetles, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae).…”

Agricultural production: assessment of the potential use of Cas9-mediated gene drive systems for agricultural pest control

“…Among these tools is a sequenced and well-annotated genome (Tribolium Genome Sequencing Consortium et al 2008), which provides the necessary sequences for Cas9 targeting. Furthermore, a number of essential genes have been identified (Lorenzen et al 2007;Trauner et al 2009;Ulrich et al 2015), and their RNAi phenotypes characterized by the iBeetle Project (Ulrich et al 2015), establishing which candidate genes are the most efficient to target for population reduction.…”