RNAi-based gene silencing in Phenacoccus solenopsis and its validation by in planta expression of a double-stranded RNA.

Abstract

Cotton is a cash crop majorly affected by many hemipteran pests, among them the cotton mealybug, Phenacoccus solenopsis. Cotton mealybug attack has a devastating effect on cotton production and causes huge yield losses. In this study, 25 potential RNA interference (RNAi) target genes were selected from the iBeetle database and a transcriptome data set for P. solenopsis. To assess the effectiveness of the selected target genes, three methods were utilized to deliver double-stranded (ds)RNA (ingestion, artificial diet bioassay and transient gene silencing). dsRNA molecules at different concentrations were fed to insects and insect mortality was recorded for each target gene. Based on the mortality data, three genes, Krüppel homologue-1, ADP-ATP/Translocase and IDGF-1, were selected for further gene expression studies using a reduced concentration of dsRNA (5 μg/ml). Of the three genes, Krüppel homologue-1 showed significantly downregulated expression (by 70.81% and 84.33%) at two different time points (8 and 14 days). An RNAi silencing construct was designed for Krüppel homologue-1 under control of the double enhancer CamV35S promoter in the plant binary vector. Significant downregulation of gene expression, by 66.69% and 81.80%, was found for Krüppel homologue-1 using transient gene silencing at the same time intervals. This work provides the first evidence for targeting the Krüppel homologue-1 gene in a hemipteran pest, P. solenopsis, using RNAi technology through oral delivery and in planta-based transient gene silencing methods. © 2020 Society of Chemical Industry.