Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V‐ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation

Abstract

SummaryIn the past two decades, chloroplast genetic engineering has been advanced to achieve high-level protein accumulation but not for down-regulation of targeted genes. Therefore, in this report, lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450) and V-ATPase dsRNAs were expressed via the chloroplast genome to study RNA interference (RNAi) of target genes in intended hosts. PCR and Southern blot analysis confirmed homoplasmy and site-specific integration of transgene cassettes into the chloroplast genomes. Northern blots and real-time qRT-PCR confirmed abundant processed and unprocessed dsRNA transcripts (up to 3.45 million copies of P450 dsRNAs/μg total RNA); the abundance of cleaved dsRNA was greater than the endogenous psbA transcript. Feeding of leaves expressing P450, Chi and V-ATPase dsRNA decreased transcription of the targeted gene to almost undetectable levels in the insect midgut, likely after further processing of dsRNA in their gut. Consequently, the net weight of larvae, growth and pupation rates were significantly reduced by chloroplast-derived dsRNAs. Taken together, successful expression of dsRNAs via the chloroplast genome for the first time opens the door to study RNA interference/processing within plastids. Most importantly, dsRNA expressed in chloroplasts can be utilized for gene inactivation to confer desired agronomic traits or for various biomedical applications, including down-regulation of dysfunctional genes in cancer or autoimmune disorders, after oral delivery of dsRNA bioencapsulated within plant cells.

The Engineered Chloroplast Genome Just Got Smarter

“…Due to the high level of chloroplast transcription, dsRNA could be synthesized in large quantities and orally delivered via bioencapsulation in plant cells to target harmful genes [42]. In two examples described below lepidopteran chitin synthase (Chi) , cytochrome P450 monooxygenase ( P450 ) and V-ATPase dsRNA made in chloroplasts were used to silence these target genes in the insect gut [10, 43]. …”

“…Recently, the RNA interference (RNAi) concept was used for the first time to engineer the chloroplast genome (Figure 2E [43]). In that study the lepidopteran chitin synthase (Chi) , cytochrome P450 monooxygenase ( P450 ) and V-ATPase, β-actin genes were used to study RNAi of target genes.…”

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…Insertion of an intron sequence as a spacer has been shown to enhance silencing efficiency (Smith et al, 2000). Transplastomic plants have recently been demonstrated as a viable means for high volume dsRNA expression (Jin et al, 2015). The higher rate of transcription in chloroplast transformants compared to transgenics is attributed to polyploidy of the plastid genetic system, which enables up to 10,000 copies of the chloroplast genome per cell (Ruiz et al, 2011).…”

“…Important aspects to be taken into account include: (i) Encoding of essential processes, as this dictates the phenotypic effects in the case of successful systemic silencing of transcript. Previous studies on ingestion-mediated RNAi knockdown in H. armigera have included genes such as JHAMT (Asokan et al, 2014(Asokan et al, , 2013, ecdysone receptor and ultraspiracle protein (involved in insect molting and metamorphosis) (Yang and Han, 2014a;Zhu et al, 2012), chitin synthase (synthesis of chitin necessary for trachea, cuticle and midgut development) (Jin et al, 2015) and molt-regulating transcription factor (regulation of metamorphosis-related gene expression) (Xiong, 2013).…”

Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths

“…Stunted larval growth was produced in H. armigera that were fed on transplastomic cotton leaves that utilized the chloroplasts to deliver double stranded RNA to effect RNA interference (Jin et al 2015). We will continue the assessment of these genes as targets for providing control in these heliothine species.…”