Characterization of DvSSJ1 transcripts targeting the smooth septate junction (SSJ) of western corn rootworm (Diabrotica virgifera virgifera)

Abstract

Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera) DvSSJ1 mRNA, a Drosophila snakeskin (ssk) ortholog, show insecticidal activity and significant plant protection from WCR damage. The gene encodes a membrane protein associated with the smooth sepate junction (SSJ) which is required for intestinal barrier function. To understand the active RNA form that leads to the mortality of WCR larvae by DvSSJ1 RNA interference (RNAi), we characterized transgenic plants expressing DvSSJ1 RNA transcripts targeting WCR DvSSJ1 mRNA. The expression of the silencing cassette results in the full-length transcript of 901 nucleotides containing a 210 bp inverted fragment of the DvSSJ1 gene, the formation of a double-stranded RNA (dsRNA) transcript and siRNAs in transgenic plants. Our artificial diet-feeding study indicates that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for DvSSJ1 insecticidal activity. Impact of specificity of dsRNA targeting DvSSJ1 mRNA on insecticidal activities was also evaluated in diet bioassay, which showed a single nucleotide mutation can have a significant impact or abolish diet activities against WCR. These results provide insights as to the functional forms of plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and information contributing to the risk assessment of transgenic maize expressing insecticidal dsRNA.

Establishing RNAi for basic research and pest control and identification of the most efficient target genes for pest control: a brief guide

“…To date, only one product, the transgenic GM-maize SmartStax PRO expressing double stranded RNA targeting Dv-snf7 and Bacillus thuringiensis (Bt) insecticidal proteins (Head et al, 2017), gained approval and awaits its launch in the United States of America [ 191 ]. Another transgenic product based on RNAi mediated knock-down of the Dv-SSJ1 gene has been tested [ 188 , 195 ]. An alternative approach is the non-transgenic sprayable RNAi, which circumvents several problems of GM crops particularly in the EU such as lacking public support, long registration processes or difficulties in the efficient generation of transgenic plants [ 196 ].…”

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

“…In this work, we aimed to adapt the viroid-based system for producing recombinant RNA in E. coli to generate the dsRNAs that are used for RNAi-based pest control. As a target, we chose D. virgifera, a relevant corn pest for which RNAi-based control has been recently demonstrated by targeting the DvSSJ1 gene [28][29][30]. To express a dsRNA homologous to a fragment of the DvSSJ1 gene in the viroid-based system, we had to create a long inverted repeat in the sequence of the vector.…”

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA inEscherichia coli

“…In this work, we aimed to adapt the viroid-based system for producing recombinant RNA in E. coli to generate the dsRNAs that are used for RNAi-based pest control. As a target, we chose D. virgifera, a relevant corn pest for which RNAi-based control has been recently demonstrated by targeting the DvSSJ1 gene [28][29][30]. To express a dsRNA homologous to a fragment of the DvSSJ1 gene in the viroid-based system, we had to create a long inverted repeat in the sequence of the vector.…”

“…virgifera smooth septate junction 1 (DvSSJ1) gene encodes a membrane protein associated with smooth septate junctions, which are required for intestinal barrier function. Ingestion by WCR larvae of dsRNA homologous to DvSSJ1 induces mRNA suppression and larval growth inhibition and mortality [28][29][30]. Using the viroid-derived system to produce recombinant RNA in E. coli, we attempted to produce the large amounts of various dsRNAs homologous to DvSSJ1 to analyze their anti-WCR activity via oral feeding of insect larvae.…”