Abstract
The whitefly Bemisia tabaci (Genn.) is a pest and vector of plant viruses to crop and ornamental plants worldwide. Using RNA interference (RNAi) to down regulate whitefly genes by expressing their homologous double stranded RNAs in plants has great potential for management of whiteflies to reduce plant virus disease spread. Using a Tobacco rattle virus-derived plasmid for in planta transient expression of double stranded RNA (dsRNA) homologous to the acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes of B. tabaci, resulted in significant adult whitefly mortality. Nicotiana tabacum L. plants expressing dsRNA homologous to B. tabaci AChE and EcR were constructed by fusing sequences derived from both genes. Mortality of adult whiteflies exposed to dsRNA by feeding on N. tabacum plants, compared to non-dsRNA expressing plants, recorded at 24-hr intervals post-ingestion for three days, was >90% and 10%, respectively. Analysis of gene expression by real time quantitative PCR indicated that whitefly mortality was attributable to the down-regulation of both target genes by RNAi. Results indicated that knock down of whitefly genes involved in neuronal transmission and transcriptional activation of developmental genes, has potential as a bio-pesticide to reduce whitefly population size and thereby decrease virus spread.
Highlights
RNA interference (RNAi) technology is based on the expression of double stranded RNA that shares nearly 100% sequence homology with a desired target gene for optimal silencing[5] and has been widely used in functional genomics studies carried out on insects[6]
RNAi mediated silencing of AChE delivered by double stranded RNA (dsRNA) ingested by Helicoverpa armigera and Blattella germanica, underscores the potency of this target for gene knock downs in insects, much as has been previously achieved with commercial insecticides[35,36]
Consensus sequences were generated from the alignments and the results revealed that both the sequences have +6 0 bp nucleotides conserved among T. vaporariorum and two B. tabaci, MEAM I species and Asia II present in Pakistan (Table S1 and Figure S1)
Summary
RNA interference (RNAi) technology is based on the expression of double stranded RNA (dsRNA) that shares nearly 100% sequence homology with a desired target gene for optimal silencing[5] and has been widely used in functional genomics studies carried out on insects[6]. RNAi is a natural mechanism present in eukaryotes that serves as a defense mechanism against viruses and is involved in the regulation of gene expression depending upon internal and external environmental conditions[7] This phenomenon in animals was first demonstrated in Caenorhabditis elegans by silencing of unc-22 gene in a specific and systemic manner[8]. Many reports have demonstrated the potential for transgenic plant-mediated pest control by expressing dsRNA homologous to genes essential for insect survival[11]. Mortality has been achieved by expressing dsRNA in transgenic tobacco plants targeting the V-ATPase A gene, resulting in a significant reduction in the whitefly population size within 8 days, post-exposure[31] Another recent report has demonstrated the potential of RNAi technology for Bemisia control by down-regulating midgut specific genes involved in osmoregulation[32]. DsRNA expression to down-regulate EcR function in adult Drosophila melanogaster, demonstrated that EcR decreased expression can perturb reproduction, courtship behavior, and stress resistance[37,38], as well as adversely affecting survival and sex-mediated signaling in adults[39]
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