Coleoptera [9,20], Diptera [21], Hemiptera [13,14], Hymenop- tera [22], Isoptera [23], Lepidoptera [12,24] and Orthoptera [25]. As RdRP orthologs are thought to be absent in most orders of the insects [11], the spontaneous amplification of RNAi is considered unlikely and the silencing effects are thought to be transient. Hence, sustained RNAi would require a continuous input of large quantities of dsRNA [13,15,17]. This limitation of the RNAi procedures could be alleviated in the case of herbivorous insects if the insects’ host plants could be transformed to express dsRNA targeting insect genes. This approach has been shown to be effective by several researchers [13,14,15,17]. Their work revealed that when insects feed on dsRNA-producing transgenic host plants, dsRNA molecules penetrate the herbivores’ gut cells and reduce the expression of the target gene by post transcriptional gene silencing. This strategy, which recommended the use of stably transformed plants for achieving gene silencing in herbivorous insects was called plant mediated RNAi (in this paper abbreviated PMRi). RNAi and PMRi in insects provide a new reverse genetics research tool which has the potential to enable ‘real time’ analysis of insect gene function during herbivorous insect-plant interac- tions, as well as a powerful new means of controlling pests. However, the process appears to be challenging to implement for one of the most specious orders of herbivorous insects, the Lepidoptera [24], and is constrained by the time and labor requirements of generating stably transformed plants. Given that microarray-based transcriptomics of insects feeding on plants have revealed a plethora of regulated genes in insect exomes, more high throughput means of PMRi are required. Pitino and colleagues suggested a transient transformation strategy against aphids based on the Agro-infiltration of Nicotiana benthamiana leaf discs, [14]; whereas, a plant-virus based RNAi technique with these qualities was suggested against nematodes [26,27]. Dubreuil et al. [26] and Valentine et al. [27] engineered the tobacco rattle virus (TRV) for dsRNA production in plants. These researchers transiently transformed N. benthamiana plants with viral-based constructs and demonstrated RNAi in nematodes feeding on these plants. Here we report the development of a similar plant-virus based dsRNA producing system (VDPS) for the silencing of lepidopteran genes. To examine the utility of VDPS against insects, we attempted to silence genes of the herbivorous insect, Manduca sexta (Sphingidae, Lepidoptera), through its native host plant, N. attenuata. This plant- herbivore system was chosen because stable as well as TRV-based transient transformation systems are well established for N. attenuata [28,29] and the trophic delivery of dsRNA has recently been reported for M. sexta [16]. Secondly, unlike Bombyx mori or Helicoverpa armigera which represent domesticated and pest insect models, respectively, M. sexta is an ecological insect model whose interactions with its host are well characterized. Therefore, the development of a VDPS for M. sexta would be a useful tool for the study of ecological interactions. As the first gene targets for silencing, we selected three midgut expressed cytochrome P450 (CYP) genes. We compared the silencing efficiency of stable PMRi and the new transient VDPS, for one of the candidate CYPs. Second, Terenius et al. stated that, ‘‘it is always a concern that based on the mechanism of gene silencing, RNAi treatments may in some cases induce off-target effects’’ [24]; considering this possibility, we examined the specificity of VDPS for the silencing of M. sexta genes and analyzed ‘‘off-target’’ effects on the expression of CYPs that share the highest sequence identity with the three targeted CYPs. In addition, we silenced N. attenuata’s four dicer (DCL) genes during PMRi to evaluate if ingestion of longer undiced dsRNA increases the silencing efficiency of the targeted insect genes. Plant Mediated RNAi Silencing Manduca Sexta Genes Results Selection of MsCYPs for silencing test In preparation for a more detailed analysis of M. sexta larvae’s remarkable ability to tolerate dietary nicotine, we found literature that reported the transcripts of three MsCYPs (CYP6B46, CYP4M1 and CYP4M3) to be up-regulated in response to dietary nicotine intake [30,31]. To identify the CYPs that were most closely related to the three target candidates CYP6B46, CYP4M1 or CYP4M3, and hence potentially at the risk of being co-silenced by the RNAi procedure, we aligned all 23 MsCYP nucleotide sequences that were available as complete coding sequences in the NCBI database to construct a phylogenetic tree (Fig. 1A). We found that CYP6B45 had the highest sequence similarity to CYP6B46 (80.2%), whereas CYP4M2 was similar to both CYP4M1 (63.5%) and CYP4M3 (55.2%). Notably, CYP4M1 and CYP4M3 shared 53.3% sequence identity with each other. Four homologous regions of .21nt were identified in the alignment of CYP6B46 and CYP6B45 that were exactly identical (+231 to +267, +415 to +437, +1306 to +1334 and +1384 to +1430). No homologous regions of .21nt identical bases could be identified in the alignments of CYP4M1, CYP4M2 and CYP4M3. CYP6B46, CYP4M1 and CYP4M3 had ORFs of comparable lengths (1524, 1515 and 1503 bp, respectively). In order to keep the length of the undiced dsRNA precursor uniform for all three genes we cloned the cDNA fragments of $300 bp from each of these genes into the VDPS vector. To accomplish this, we analyzed these sequences for the availability of primer binding sites $300 bp apart from each other and selected regions to be cloned in each candidate cDNA. The similarity of this selected $300 bp region from CYP6B46 with its homolog in CYP6B45 was 80.4%. This region contained one .21nt (+415 to +437 of the ORF = +112 to +134 of selected $300 bp fragment) stretch that was identical in the two aligned fragments (Fig. S1A). The $300 bp regions selected from CYP4M1 and CYP4M3 were homologous to each other (54.1% similar), whereas their similarity with the homologous region from CYP4M2 was 64.5% and 57.1%, respectively (Fig. S1B). The exact sizes of these selected regions were 312 bp (+301 to +612), 338 bp (+1000 to +1337) and 322 bp (+966 to +1287) in CYP6B46, CYP4M1 and CYP4M3, respectively. PMRi is thought to mainly target genes that are expressed in gut tissues [13,15,17]. Therefore, to ascertain whether the candidate genes (CYP6B46, CYP4M1 and CYP4M3) were gut expressed, we profiled their transcripts along with the transcripts of the allied co- target (CYP6B45 and CYP4M2) genes in hemolymph, Malpighian tubules, fat body, foregut, midgut and hindgut. All five genes were found to have relatively higher expression levels in the gut regions as compared to the other tissues (p#0.05; Fig. 1B). The primers used for this profiling specifically amplified respective insect cDNA; they did not produce any amplicons when plant cDNA was used as a PCR template (Fig. S1C). Stable transgenic plant mediated RNAi (PMRi) for MsCYP6B46 We generated stable transgenic lines of N. attenuata plants transformed with a pSOL8 vector harboring an inverted repeat (ir) of the selected 312 bp CYP6B46 cDNA fragment (Fig. S2A). A single insertion of the transgene was confirmed by southern hybridization, in two T2 generation lines (ir-CYP6B46 30-2 and ir- CYP6B46 416-3) that were generated from two independent transformation events (Fig. S2B). Transcription of 312 bp CYP6B46 ‘ir’ insertions and the subsequent formation of diced PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31347