Plant Mediated RNAi Silencing Manduca Sexta Genes Figure 1. Selection of M. sexta CYPs for plant mediated RNAi (PMRi) and their spatial expression profiles. (A) Phylogenetic relationship among M. sexta CYPs (complete ORFs) as calculated by the Clustal-W program. CYPs selected for silencing by PMRi are shaded in gray and their most closely related CYPs that were analyzed for off-target co-silencing are in dashed boxes. Thousand bootstrapping trials were conducted (only the bootstrap values .50 displayed). Transcript levels (relative to ubiquitin) of (B) CYP6B46, (C) CYP6B45, (D) CYP4M1, (E) CYP4M3 and (F) CYP4M2 in hemolymph, Malpighian tubules, fat body, foregut, midgut and hindgut of 5th instar M. sexta larvae feeding on N. attenuata (WT) plants. Bars labeled with different letters indicate the significant differences as determined by one way ANOVAs (p#0.05). doi:10.1371/journal.pone.0031347.g001 small dsRNA (21–24nt) (smRNA) in the leaves of the ir-CYP6B46 lines were ascertained by northern hybridization (Fig. 2A). CYP6B46 smRNA was not detected in the leaves of controls wild type (WT) and empty pSOL8 vector (EV) plants. Morphology and development of the plants of both ir-CYP6B46 lines were similar to those of WT or EV plants. Freshly hatched neonates of M. sexta larvae were placed on control and ir-CYP6B46 (30-2 and 416-3) lines and allowed to feed freely for 14 days. Using Northern hybridization, we confirmed the presence of CYP6B46 smRNA in their midgut (Fig. 2A). No CYP6B46 smRNA could be detected in the midguts of larvae that fed on the WT and EV plants (Fig. 2A). Further, CYP6B46 transcript levels in the midgut of these 14 d old larvae feeding on two ir-CYP lines were significantly reduced (three fold) compared to those of the larvae feeding on WT or EV plants (p#0.05; Fig. 2B). Interestingly, the silencing of this gene had no effect on larval mass gain recorded after 14 d of feeding (p.0.05; Fig. 2D). PMRi mediated silencing was target gene specific To ascertain if the silencing was specific to the target gene we quantified the transcript levels of the closely related off-target gene, CYP6B45. The transcript levels of CYP6B45 in the midguts of larvae feeding on WT, EV and transgenic ir-CYP6B46 (30-2 and 416-3) plants did not differ (p.0.05; Fig. 2C). VDPS mediated silencing of MsCYP6B46, MsCYP4M1 and MsCYP4M3 In order to rapidly and efficiently synthesize the dsRNA in planta, virus induced gene silencing (VIGS) vector pTV containing the antisense cDNA fragment ($300 bp) of MsCYP6B46, MsCYP4M1 or MsCYP4M3 (Fig. S3A) was Agro-infiltrated into N. attenuata WT leaves, as described by Saedler and Baldwin [29]. The smRNA produced in the leaves was detected by Northern hybridization, using the appropriate gene specific probe (Fig. 3A– PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31347