Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/26/2007
Publication Date: 9/1/2007
Citation: Sukno, S.A., Mccuiston, J., Wong, M., Wang, X., Thon, M.R., Hussey, R., Baum, T., Davis, E. 2007. Quantitative Detection of Double-Stranded RNA-Mediated Gene Silencing of Parasitism Genes in Heterodera glycines. Journal of Nematology. 39:145-152. Interpretive Summary: Plant parasitic nematodes cause significant economic damage to many agricultural crops. In order to successfully parasitize a root, nematodes secrete proteins that transform plant cells into a specialized site for the nematode to feed. Understanding the function of nematode parasitism genes encoding these secreted proteins will be very useful for identifying nematode targets for novel control strategies. Currently, RNA interference (RNAi) has emerged as a powerful tool for assessing gene function. In this study, we have improved the current procedure for RNAi silencing of nematode genes by the nematode soaking method. The new procedure developed provides a more effective tool in evaluating the function of nematode parasitism genes that may be targeted for generating novel control strategies.
Technical Abstract: The introduction of a double-stranded RNA (dsRNA) into an organism to induce sequence-specific RNA interference (RNAi) of a target transcript has become a powerful technique to investigate gene function in nematodes and many organisms. Data provided here indicate that the inclusion of 1-2 mM spermidine and 50 mM octopamine and a 24 hr incubation period of nematodes in double-stranded RNA (dsRNA) soaking solutions resulted in a considerable increase in the percentage of nematodes that ingested dsRNA as compared to previous reports. This modified dsRNA soaking method was coupled with quantitative real-time RT-PCR (qRT-PCR) analyses to assess the potential silencing of the Heterodera glycines parasitism gene transcripts Hg-pel-1 and Hg-4E02 that are expressed within the esophageal gland cells of preparasitic H. glycines J2. The Hg-pel-1 transcript was most efficiently silenced with one dsRNA construct (ds267) at the highest dsRNA soaking concentration of 5.0 mg/ml, while the Hg-4E02 transcript was more efficiently silenced at the 2.5 mg/ml dsRNA concentration as compared to 5.0 mg/ml. A dsRNA construct (ds285) complementary to a different sequence within the Hg-pel-1 transcript than construct ds267 induced only minimal silencing of the Hg-pel-1 transcript at 2.5 mg/ml. The results suggest that both dsRNA concentration and sequence relative to the transcript targeted are critical for maximizing potential RNAi effects in parasitic nematodes.