Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe

Abstract

To control Heterodera glycines Ichinohe (soybean cyst nematode) in Glycine max (L.) Merr. (soybean), we evaluated the use of producing transgenic soybean seedlings expressing small interfering RNAs (siRNAs) against specific H. glycines genes. Gene fragments of three genes related to nematode reproduction or fitness (Cpn-1, Y25 and Prp-17) were PCR-amplified using specific primers and independently cloned into the pANDA35HK RNAi vector using a Gateway cloning strategy. Soybean roots were transformed with these constructions using a composite plant system. Confirmation of transformation was attained by PCR and Southern blot analysis. Transgene expression was detected using reverse transcription PCR (RT-PCR) and expression of siRNAs was confirmed in transgenic plants using northern blot analysis. Bioassays performed on transgenic composite plants expressing double-stranded RNA fragments of Cpn-1, Y25 and Prp-17 genes resulted in a 95, 81 and 79% reduction for eggs g(-1) root, respectively. Furthermore, we demonstrated a significant reduction in transcript levels of the Y25 and Prp-17 genes of the nematodes feeding on the transgenic roots via real-time RT-PCR whereas the expression of non-target genes were not affected. The results of this study demonstrate that over-expression of RNA interference constructs of nematode reproduction or fitness-related genes can effectively control H. glycines infection with levels of suppression comparable to conventional resistance.

Co-regulation of the Glycine max soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-containing regulon occurs during defense to a root pathogen

“…All of the experiments presented here at least meet and in most cases exceed these published standards (Golden et al 1970;Riggs & Schmidtt 1988Kim et al 1998;Niblack et al 2002). The FI assay is also the community-accepted standard analysis method used in experiments in other labs employing genetically engineered constructs in G. max, including those using K599, to examine H. glycines biology (Mazarei et al 2007;McLean et al 2007;Steeves et al 2006;Li et al 2010;Melito et al 2010;Liu et al 2011Liu et al , 2012Matthews et al 2013Matthews et al , 2014. Following the published methods employed in those studies, in the analysis presented here Nx is the pRAP15-GOI or pRAP17-GOI-transformed line and Ns is the pRAP15-ccdB or pRAP17-ccdB control.…”

Host-Derived Artificial MicroRNA as an Alternative Method to Improve Soybean Resistance to Soybean Cyst Nematode

“…Nematode bioassays on transgenic hairy roots could always generate variable results depending on the transformation and expression levels of each construct in different events, as well as the condition of nematode culture [42,43]. The mixed transgenic hairy root (as shown in Figure 4) could also affect the SCN survival rate.…”

Advancements in Transgenic Soy: From Field to Bedside

“…RNA silencing was elicited in the cyst nematode following ingestion of dsRNA molecules, and resulted in ~75% suppression of reproductive capabilities. Several years later Li et al, used RNAi to test potential gene targets known to be involved with nematode reproduction and fitness [36]. Soybean roots expressing small interfering RNAs against the SCN genes Cpn-1, Y25, and Prp-17 showed a significant reduction in transcript levels in nematode feeding sites.…”