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Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte).
PLoS One. 2012; 7(10):e47534.Plos

Abstract

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.

Authors+Show Affiliations

Biotechnology Division, Monsanto Company, Chesterfield, MO, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

23071820

Citation

Bolognesi, Renata, et al. "Characterizing the Mechanism of Action of Double-stranded RNA Activity Against Western Corn Rootworm (Diabrotica Virgifera Virgifera LeConte)." PloS One, vol. 7, no. 10, 2012, pp. e47534.
Bolognesi R, Ramaseshadri P, Anderson J, et al. Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). PLoS One. 2012;7(10):e47534.
Bolognesi, R., Ramaseshadri, P., Anderson, J., Bachman, P., Clinton, W., Flannagan, R., Ilagan, O., Lawrence, C., Levine, S., Moar, W., Mueller, G., Tan, J., Uffman, J., Wiggins, E., Heck, G., & Segers, G. (2012). Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). PloS One, 7(10), e47534. https://doi.org/10.1371/journal.pone.0047534
Bolognesi R, et al. Characterizing the Mechanism of Action of Double-stranded RNA Activity Against Western Corn Rootworm (Diabrotica Virgifera Virgifera LeConte). PLoS One. 2012;7(10):e47534. PubMed PMID: 23071820.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte). AU - Bolognesi,Renata, AU - Ramaseshadri,Parthasarathy, AU - Anderson,Jerry, AU - Bachman,Pamela, AU - Clinton,William, AU - Flannagan,Ronald, AU - Ilagan,Oliver, AU - Lawrence,Christina, AU - Levine,Steven, AU - Moar,William, AU - Mueller,Geoffrey, AU - Tan,Jianguo, AU - Uffman,Joshua, AU - Wiggins,Elizabeth, AU - Heck,Gregory, AU - Segers,Gerrit, Y1 - 2012/10/11/ PY - 2012/08/16/received PY - 2012/09/12/accepted PY - 2012/10/17/entrez PY - 2012/10/17/pubmed PY - 2013/2/23/medline SP - e47534 EP - e47534 JF - PloS one JO - PLoS One VL - 7 IS - 10 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/23071820/Characterizing_the_mechanism_of_action_of_double_stranded_RNA_activity_against_western_corn_rootworm__Diabrotica_virgifera_virgifera_LeConte__ L2 - https://dx.plos.org/10.1371/journal.pone.0047534 DB - PRIME DP - Unbound Medicine ER -