Plant Mediated RNAi Silencing Manduca Sexta Genes Figure 3. Efficiency and specificity of CYP6B46, CYP4M1 and CYP4M3 silencing by viral dsRNA-producing system (VDPS). Northern hybridization showing the smRNAs of (A) CYP6B46 (B) CYP4M1 and (C) CYP4M3 in WT leaves infiltrated with VDPS-EV, -CYP6B46, -CYP4M1 and - CYP4M3 constructs, respectively, as well as in the midguts of 4th instar larvae feeding on the leaves of plants inoculated with the respective constructs. Lane M shows 21 b oligonucleotide that was used as size marker as well as a positive control for hybridization. Similar fluorescence intensity of the ethidium bromide stained 5.8 S rRNA bands reflected the equal loading of LMW RNA. Transcript abundance (relative to ubiquitin) of the target genes. (D) CYP6B46 in the midguts of 4th instar larvae feeding on VDPS-EV and -CYP6B46, (F) CYP4M1 in the midguts of 4th instar larvae feeding on VDPS-EV, -CYP4M1 and -CYP4M3, and (H) CYP4M3 in the midguts of 4th instar larvae feeding on VDPS-EV, -CYP4M3 and -CYP4M1 plants, respectively. Transcript abundance (relative to ubiquitin) of closely related, off-target genes (E) CYP6B45 in the midguts of 4th instar larvae feeding on VDPS-EV and -CYP6B46, (G) CYP4M2 in the midguts of 4th instar larvae feeding on VDPS-EV and -CYP4M1, and (I) CYP4M2 in the midguts of 4th instar larvae feeding on VDPS-EV and -CYP4M3 plants, respectively. (J) Mass of 4th instar M. sexta larvae fed for 14 days on VDPS-EV, -CYP6B46, -CYP4M1 and -CYP4M3 plants. Asterisk indicates the significant differences as determined by one way ANOVAs (p#0.05). doi:10.1371/journal.pone.0031347.g003 strating that the silencing the plant’s dicer machinery had increased the silencing efficiency of the PMRi by a factor of 2. Discussion PMRi has the potential of becoming a crop protection tool targeting insect pests with far greater specificity than currently available pesticides or xenobiotics such as the Bt toxin [15]. Especially in the case of lepidopteran insects, CYPs that detoxify plant defense compounds or synthetic insecticides are potential targets of RNAi. Silencing these CYPs, suppresses transcript levels of the targeted genes, attenuating their function, and influencing larval growth or survival [15,17,33]. Since they belong to multigene families, CYPs have also been proposed to be the ideal targets for combinatorial RNAi [17]. Therefore in this first attempt of insect gene silencing by transient plant viral system, we considered CYPs as valuable candidate genes. Snyder and colleagues demonstrated that CYP4M1 and CYP4M3 were upregulated in M. sexta larval midgut in response to nicotine ingestion [31]. Similarly in a microarray analysis, Govind and colleagues showed that CYP6B46 was downregulated in the larval midgut, when fed on nicotine suppressed ir-PMT plants [30]. Based on their response to dietary nicotine, we selected these three genes (CYP6B46, CYP4M1 and CYP4M3) for the PMRi trial. M. sexta is the specialist herbivore of host plants that produce high concentrations of nicotine and is famous for having the highest tolerance to nicotine of any organism [34]. Several researchers demonstrated a successful insect gene silencing with the Agrobacterium-transformed stable or transient transgenic plant mediated RNAi [13,14,15,17]. We report similar silencing for the midgut-based CYP6B46 of M. sexta, suggesting that the PMRi is a reproducible and robust technique. Additionally, we showed that the silencing was highly specific and did not spread even to the most similar genes of the same PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31347