Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference Pavan Kumar, Sagar Subhash Pandit, Ian T. Baldwin* Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Jena, Germany Abstract Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA. Conclusions: Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect’s RNAi machinery is more efficient with longer lengths of ingested dsRNA. Citation: Kumar P, Pandit SS, Baldwin IT (2012) Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference. PLoS ONE 7(2): e31347. doi:10.1371/journal.pone.0031347 Editor: Luciano A. Moreira, Centro de Pesquisas Rene´ Rachou, Brazil Received October 12, 2011; Accepted January 6, 2012; Published February 1, 2012 Copyright: 2012 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Max Planck Society. PK was funded by a DAAD PhD fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: baldwin@ice.mpg.de Introduction RNA interference (RNAi), the double stranded RNA (dsRNA) mediated gene silencing was discovered in nematodes in 1998 [1]. During the RNAi process, dsRNA produced in the nucleus is transported to the cytoplasm; alternatively, exogenous dsRNA can be taken up by cells with the help of the cell surface protein, SID [from systemic RNAi deficient mutants (sid)] [2]. In the cytoplasm, dsRNA is cleaved by RNaseIII type enzymes (dicers) to produce approximately 22 bp fragments, called small interfering RNAs (siRNAs) [3,4]. One strand of the siRNA (guide strand) is incorporated into the RNA-induced silencing complex (RISC) with the perfectly complementary site in a target mRNA to form a guide strand-target mRNA duplex [5]. The target mRNA is then sliced by the Argonaute protein of RISC [6]. In plants and nematodes, RNAi is amplified by the activity of RNA-dependent RNA polymerases (RdRPs). This enzyme extends the guide strand that is bound to the target mRNA towards its 59 end [3,7]. The long dsRNA formed in this process re-enters the RNAi cycle after it is cleaved by dicers. RNAi-mediated gene silencing becomes a systemic process as the siRNAs spread to neighboring cells to induce a fresh cycle of dicing and splicing Mlotshwa1 [3,7,8]. This endogenous gene silencing mechanism has been exploited as a reverse genetic tool for several model organisms and has also been proposed as a potentially useful tool for pest control [9,10]. However, there are two major limitations to the widespread use of RNAi in insects: 1) the generation of stably transformed lines of insects for the reverse genetics research is onerous, and 2) the apparent lack of key components of the RNAi pathway in insects, namely SIDs and RdRPs [11], which requires that large quantities of triggering siRNAs be continuously administered to sustain gene silencing. To understand if RNAi is functional in insects, researchers have delivered dsRNA to insects by various methods: feeding [12,13,14,15,16,17], injection [18] and exogenous appli- cation [19]. Interestingly, the success of silencing genes by these different delivery modes differs amongst the various insect orders: PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31347