Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation

Summary

In the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation  but  not  for  down-regulation  of  targeted  genes. Therefore, in this report, lepidopteran chitin synthase (Chi),  cytochrome  P450  monooxygenase  (P450)  and  V-ATPase  dsRNAs  were  expressed  via  the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis  confirmed  homoplasmy  and  site-specific  integration  of  transgene  cassettes  into  the  chloroplast genomes.  Northern  blots  and  real-time  qRT-PCR  confirmed  abundant  processed  and  unprocessed dsRNA transcripts  (up  to  3.45  million  copies  of  P450  dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater  than  the  endogenous  psbA  transcript.  Feeding  of  leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to  almost  undetectable  levels  in  the  insect midgut, likely after further processing of dsRNA in their gut.  Consequently, the  net  weight  of  larvae,  growth  and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most  importantly,  dsRNA  expressed  in chloroplasts  can  be  utilized  for  gene inactivation to confer desired agronomic traits  or  for  various  biomedical  applications,  including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522700/?report=classic