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Using plants to silence insect genes in a high-throughput manner
Date:2/2/2012

uences of CYP genes in Manduca sexta and were able to switch off these genes using RNAi technology, but expressed in the plant.

Using plants to silence insect genes

RNA interference (RNAi) is triggered by the production of double-stranded RNA (dsRNA) comprising about 300 base pairs in the cells of tobacco plants. If larvae feed on these plant, the RNA is released in the insect gut. In the experiments, the dsRNA harbored the sequence of the insect gene,CYP6B46, a special cytochrome P450 oxidoreductase specific for Manduca sexta larvae. In a next step, the dsRNA was enzymatically broken down into smaller RNA segments; a special enzyme complex called RISC (RNA-induced silencing complex), which carries several of these RNA segments, specifically binds to the messenger RNA (mRNA) of the CYP6B46 gene and disassembles the mRNA in such a way that the cytochrome P450 enzyme cannot be produced anymore. "We were impressed by the high specificity of these RNAi experiments. The analysis of mRNA transcripts of closely related CYP6 genes revealed that only the CYP6B46 gene was silenced. This means that there was no collateral damage from the procedure: the gene silencing worked on only one targeted gene," says Ian Baldwin.

The use of additional CYP RNAi probes revealed further interesting results: Young caterpillars which had ingested dsRNA of the CYP4M3 gene gained significantly less weight within 14 days in comparison to larvae reared on control plants very likely a consequence of the nicotine and its toxic effect which had been restored by switching off the CYP gene. The RNAi experiments had been conducted using plant viral vectors. Unlike genetically transformed tobacco plants in which CYP dsRNA is produced constitutively, the virus vector-based technique provides dsRNA transiently produced in wildtype tobacco plants. Both methods worked well but the "plant virus-based dsRNA producing system" (VDPS) allows for a throughput of RN
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Contact: Ian T. Baldwin
baldwin@ice.mpg.de
49-364-157-1101
Max Planck Institute for Chemical Ecology
Source:Eurekalert  

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