Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte).
RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.
Biotechnology Division, Monsanto Company, Chesterfield, MO, USA.
Renata
R
Bolognesi
Parthasarathy
P
Ramaseshadri
Jerry
J
Anderson
Pamela
P
Bachman
William
W
Clinton
Ronald
R
Flannagan
Oliver
O
Ilagan
Christina
C
Lawrence
Steven
S
Levine
William
W
Moar
Geoffrey
G
Mueller
Jianguo
J
Tan
Joshua
J
Uffman
Elizabeth
E
Wiggins
Gregory
G
Heck
Gerrit
G
Segers
D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D015153:Blotting, Western; D001517:Coleoptera / Q000187:drug effects* / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D041981:Gastrointestinal Tract / Q000378:metabolism; D007300:Insect Control / Q000379:methods*; D007814:Larva / Q000187:drug effects; D007928:Lethal Dose 50; D008969:Molecular Sequence Data; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000633:toxicity*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D056827:Endosomal Sorting Complexes Required for Transport; D012330:RNA, Double-Stranded