p406TEF1

RNA interference has made significant progress as a developmental tool for pest management. In laboratory experiments, double-stranded RNA (dsRNA) is often administered to insects through genetic modification of crops, or is synthesized in vitro and applied topically to crops. Here, researchers engineered transgenic yeast to express dsRNA targeting y-Tubulin in Drosophila suzukii. Yeast grows naturally on the surface of fruit crops and is a major component of the fruit fly microbiome, making it highly attractive to fruit flies. Therefore, this naturally attractive yeast biopesticide can deliver dsRNA to pests without the need for genetic modification of the crop. The study demonstrated that this biopesticide reduced larval survival and reduced locomotor activity and reproductive fitness of adults, indicating a decrease in overall health.

In this study, DNA plasmids were constructed to constitutively express a dsRNA hairpin targeting D. suzukii key genes y-tubulin 23C (yTub23C) (183 bp), bellwether (blw) (230 bp), Ribosomal protein L19 (RpL19) (215 bp) and acetylcholinesterase (Ace) (221 bp). Approximately 200 bp of the Drosophila suzukii target gene sequence was inserted into a DNA plasmid to form an inverted repeat and joined by a 74 bp intron sequence from the white gene of Drosophila melanogaster. This plasmid is self-replicating and does not integrate into the host yeast genome, so the host genome remains unchanged. The plasmid was transformed into S. cerevisiae strain INVSc1 using heat shock and expression of dsRNA was verified using RT-qPCR. The empty P406TEF1 plasmid was transformed into S. cerevisiae strain INVSc1 and used as a control strain. The researchers chose to use a yeast strain containing an empty plasmid and not expressing dsRNA as a control strain to avoid possible off-target effects, such as inhibition of key genes that may have regions of sufficient homology to the control sequence to generate an RNAi response.

Figure 1. (a) Map of the transformation vector used to express hairpin dsRNA in Saccharomyces cerevisiae. Approximately 200 bp of the D. suzukii target gene sequence was inserted into p406TEF1, forming an inverted repeat connected by a 74 bp intronic sequence from the white gene. (b) Expression of dsRNA in Saccharomyces cerevisiae. Expression was quantified using RT-qPCR and normalized to the housekeeping gene actin. The empty P406TEF1 plasmid was transformed into S. cerevisiae strain INVSc1 and used as a control strain. (Murphy K A, et al., 2016)