Limitations of RNA interference as a potential technique for crop protection against insect pests

Abstract

The RNAi response suppresses gene expression at the post-transcriptional level and the potential of this technique to give control of insect pests in crops has been recognised for a decade.
This project focuses on a comparison of RNAi responses in insects of different orders by injecting and feeding dsRNA directed against thread (APIN) and V-Type- ATPase E homologues in the target species. The results showed systemic RNAi responses, and mortality, occurred in larvae of the Tribolium castaneum (T. castaneum), Coleopteran, but not a Hemipteran, the Acyrthosiphon pisum (A. pisum), where comparatively low levels of gene down-regulation were only achieved by injection of dsRNA. DsRNA injection produced both a lethal phenotype and gene down- regulation in larvae of the dipteran species Musca domestica (M. domestica), and Delia radicum (D. radicum), although the effects were found to be stage dependent.
Rapid dsRNA degradation in the extracellular environment could lead to a limitation of RNAi reponses. In vitro experiments show that dsRNA was degraded rapidly by A. pisum haemolymph and gut extracts, and less rapidly by D. radicum larval extracts. However, T. castaneum larval extracts differ in both the amount and qualitative nature of their RNase activity; dsRNA was degraded at a slow rate, predominantly by exonuclease activity rather than endonuclease activity.
A strategy using recombinant proteins was used to address limitations of RNAi effects after feeding dsRNA in insects. A recombinant protein containing an RNA binding domain (RBD) was selected to conjugate dsRNA forming a protein-RNA complex. The complexed protein enhanced the stability of dsRNA and protected it from degradation from insect extracts. A fusion protein containing snowdrop lectin (GNA) linked to RBD was also developed to produce a "systemic" RNAi effect, by transporting the protein-RNA complex to the insect haemolymph using the lectin as a "carrier".