Successful PMRi in M. sexta allowed us to use it as benchmark for developing an easier and faster VDPS method. While trying the VDPS that was previously successful against root nematodes [26,27], we found that the silencing efficiency of VDPS and PMRi was similar. The VDPS experiments could be accomplished within three months in contrast to the PMRi procedure that requires a year of laborious screening of plants through two generations. Since VDPS is a more rapid technique, we could screen three CYPs in a short time and found CYP4M3 to be good candidate for further research. Since the larval growth was reduced while feeding on nicotine containing VDPS-CYP4M3 plants (Fig. 3J), this gene among the three CYPs tested, may play a central role in increasing M. sexta’s tolerance to nicotine ingestion. Similar to stable PMRi, VDPS was also found to be highly specific in its silencing of all three candidate genes. PMRi is clearly the method of choice for crop protection in countries which allow the growth of transgenic crops and could be of immediate utility in the control of polyphagous pests such as H. armigera. However, VDPS could be a method of choice for high throughput reverse genetic screens of potential genetic targets in insect pests, as well as enabling research into a multitude of unanswered ecological questions at the molecular level. The length of dsRNA is often of concern in RNAi experiments. In most of the experiments on insects that were based on the trophic delivery of dsRNA, different lengths of dsRNA (300– 520 bp) has been used [2]. In the TRV based VDPS used against nematodes, inserts greater than 150 bp were recommended [27]. However, cloning of small sense or antisense fragments and especially small hairpins were also shown to be effective in the TRV vectors [27,35]. Therefore the standardization of insert length would be an important consideration for the future VDPS experiments. We addressed the issue of dsRNA length in the PMRi experiment. Silencing of CYP6B46 was enhanced after increasing the length of ingested dsRNA by silencing the plant’s dicers. These results were consistent with the findings in H. armigera [15] and are consistent with the hypothesis that the efficiency of RNAi depends on the length of the ingested dsRNA. Recent discoveries have shown that the dsRNase from B. mori midgut is synthesized in midgut cells and subsequently secreted into the lumen [36,37,38]. Thus, it is possible that the lepidopteran dicers that function in extremely alkaline environments of the midgut are specialized and possess different dicing properties than do the plant dicers; consequently, insect-dicer diced smRNA might be more effective than the plant-dicer diced smRNA in gene silencing in insects. We conclude that stable PMRi can be a specific and robust system of gene silencing in M. sexta. While retaining all the virtues of PMRi, VDPS promises to be a rapid and high throughput alternative, suitable for ecological research. Silencing of plant dicers in PMRi lines revealed that similar to the results obtained in H. armigera, the gene silencing effect is enhanced in M. sexta suggesting that plant and insect RNAi machinery respond differently to the varying lengths of dsRNA. Materials and Methods Plant material N. attenuata 306 inbred seeds, which were originally collected in 1988 from a native population at Utah (United States) were used for the generation of Agrobacterium tumefaciens mediated stable transgenic as well as VDPS lines in all the experiments. The seeds were germinated on sterile Gamborg B5 medium (Sigma, Germany) after 1 h of treatment with 506 (V/V) diluted smoke (House of Herbs) and 1 mM GA3. Ten days after germination, Plant Mediated RNAi Silencing Manduca Sexta Genes seedlings were transferred to Teku pots containing peat-based substrate, and after an additional 10 to 12 d, the plantlets were transplanted into individual 1L pots with the same substrate. In the glasshouse, plants were grown at 24uC to 26uC, relative humidity approximately 55%, and supplemented with light from 400- and 600-W sodium lamps (Philips) for 16 h [39]. Insect culture Eggs from an in-house M. sexta colony were stored in a growth chamber (Snijders Scientific) at 26uC- 16 h light, 24uC- 8 h darkness and 65% relative humidity, until the larvae hatched. Three freshly hatched neonates were placed on each rosette-stage N. attenuata plant. Fourteen day old fourth instar larvae were used in all assays, unless specified otherwise. Analysis of MsCYP sequences and their selection for cloning in PMRi or VDPS vector Sequences of 23 M. sexta CYPs were retrieved as complete coding sequences from NCBI. Their ORFs were aligned and a phylogenetic tree was constructed using Clustal W, with 1000 bootstrapping trials, to identify the most closely related MsCYPs to the preselected targets of gene silencing: CYP6B46 (GU731529), CYP4M1 (GU731525) and CYP4M3 (GU731527). Sequences of candidate gene cDNA fragments ($300 bp) to be cloned in the PMRi or VDPS vector were selected based on two criteria: 1) the availability of primer binding sites $300 bp apart from each other in the candidate cDNA, according to the ‘Primer3’ online utility [40] and 2) the degree of similarity of this $300 bp fragment with their homologous region in the allied genes. This was considered in order to avoid co-silencing of closely related genes due to their high sequence similarity and to enable detection of ‘off-target’ effects of gene silencing [24]. Thus, CYP6B46 fragment was aligned with its homolog from CYP6B45 (GU731528), and the CYP4M1 and CYP4M3 fragments (which are already highly homologous to each other) were aligned with their closest homolog, CYP4M2 (L38671) (Fig. S1A, S1B). Larval tissues Hemolymph, Malpighian tubules, fat body, foregut, midgut and hindgut were collected from the fifth instar larvae for transcript profiling of the selected MsCYPs. Hemolymph of each larva was collected separately by clipping the horn. Then, these larvae were dissected in 0.15 M NaCl under a dissecting microscope. The 14 d old fourth instar larvae used in all gene silencing assays were also dissected in 0.15 M NaCl to isolate their midguts. Dissected gut tissues were carefully washed in 0.15 M NaCl to remove any adhering plant material. All tissues were stored in TRI regent (Invitrogen) as recommended by the manufacturer, until further use. Total RNA isolation Total RNA was extracted from TRI regent stored tissues using manufacturer’s protocol. Isolated total RNA was always subjected to TURBO DNase (Ambion) treatment, according to the manufacturer’s protocol. Real time quantitative PCR All the primer sequences used for real time quantitative PCR (qRT-PCR) are listed in Table S1. These primers were designed from the transcript sequences retrieved from NCBI, using Primer3 [40], to amplify $100 bp in the respective template cDNA, upstream of the selected $300 bp regions. This upstream position was targeted because, in the organisms such as plants, nematodes and fruit flies in which the RNAi pathway has been well PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31347