Figure 2. PMRi of M. sexta CYP6B46 using stably transformed N. attenuata plants. (A) Northern hybridizations revealed the presence of small RNAs of CYP6B46 in the leaves of two independent lines of stably transformed plants, ir-CYP6B46 (30-2) and ir-CYP6B46 (416-3), and in the midguts of 4th instar larvae feeding on these plants. RNA samples from leaves of WT and EV (empty vector transformed stable line) plants and from the midguts of larvae feeding on WT and EV leaves were used as negative controls. Similar fluorescence intensity of the ethidium bromide stained 5.8 S rRNA bands reflected the equal loading of LMW RNA. Low molecular weight RNA from leaf or midgut loaded on the gel in each lane was a pool of three biological replicates. smRNA length of 21 b denoted by marker. Transcript abundance (relative to ubiquitin) of: (B) CYP6B46 (target gene) and closely related (C) CYP6B45 (off-target) in the midguts of 4th instar larvae. (D) Larval mass of 4th instar larvae feeding on WT, EV, ir-CYP6B46 (30-2) and ir-CYP6B46 (416- 3) N. attenuata plants for 14 days. Asterisk indicates the significant differences as determined by one way ANOVAs (p#0.05). doi:10.1371/journal.pone.0031347.g002 3C). For all three CYPs, the negative control leaves (EV) did not contain any of the target smRNAs. Freshly hatched M. sexta neonates were transferred to VDPS- CYP6B46, -CYP4M1, -CYP4M3 and -EV plants. After 14 d of feeding, the midguts of these larvae were analyzed for the presence of the respective smRNAs by Northern hybridization. The expected smRNAs were detected in the midguts of larvae feeding on VDPS-CYP6B46, -CYP4M1 and -CYP4M3 lines (Fig. 3A– 3C). The success of the silencing of the target gene was quantified Plant Mediated RNAi Silencing Manduca Sexta Genes by the reduction in the transcript levels of the target gene in the midguts of larvae feeding on the respective VDPS-CYP line, compared to larvae feeding on VDPS-EV. VPDS reduced CYP6B46 transcripts by three fold (Fig. 3D), reductions that were equivalent to that mediated by PMRi (Fig. 2B). Transcripts of CYP4M1 and CYP4M3 were reduced by 50% in the midguts of larvae feeding on the respective VDPS plants, as compared to that in the midguts of EV fed larvae (p#0.05; Fig. 3F, 3H). Larval performance measured in terms of body mass after feeding for 14 d on VDPS-CYP6B46 and VDPS-EV plants was unchanged (p.0.05; Fig. 3J): a result congruent with the larval mass gain observed on stable PMRi lines (ir-CYP6B46 and EV) (Fig. 2D). Larvae feeding on VDPS-CYP4M1 plants also did not show any difference in body mass as compared to that of larvae feeding on control EV plants (p.0.05; Fig. 3J). However, the larvae feeding on VDPS-CYP4M3 plants gained significantly less mass compared to EV fed larvae (p#0.05; Fig. 3J). VDPS mediated silencing is target gene specific Levels of CYP6B45 transcripts, the off-target gene, remained unchanged in the midguts of larvae feeding on EV or VDPS- CYP6B46 plants (p.0.05; Fig. 3E), suggesting that the CYP6B46 silencing attained by VDPS was comparably specific to that attained by stable PMRi (Fig. 2C). Similarly, the silencing of CYP4M1 did not cause co-silencing of CYP4M3 and vice versa (p.0.05) (Fig. 3F, 3H); moreover, larvae feeding on these two VDPS lines (-CYP4M1 and -CYP4M3) had similar CYP4M2 transcript levels in their midguts (compared to larvae feeding on EV; p.0.05) (Fig. 3G, 3I). Silencing of plant dicers enhances the silencing of CYP6B46 by PMRi DCLs are involved in the biogenesis of smRNA by cleaving longer dsRNA. Four different types of DCLs are reported in higher plants. Their function has been found to overlap in plants, suggesting that one DCL can contribute to and/or compensate for the function of the others. Hence, more than one DCL might be involved in processing long dsRNA [32]. Efficiency of plant mediated silencing of H. armigera gene was previously shown to be increased by feeding long dsRNA that was obtained by silencing three Arabidopsis dicers 2, 3 and 4 [15]. We aimed to understand if the plant dicers functioned similarly in the N. attenuata- M. sexta model. To select the most effective combination of DCLs to silence, so as to increase the concentration of the retained longer dsRNA, we Agro-infiltrated all 16 combinations of the four NaDCL constructs into the stable PMRi line ir-CYP6B46 (30-2) (Fig. 4A). We found that silencing NaDCL 4, or the simultaneous silencing of any three NaDCLs significantly increased the accumulation of long dsRNA (102 bp that could be quantified by qRT-PCR) in plant leaves (p#0.05; Fig. 4B, S4A). We selected the combinations of NaDCL 1, 3 and 4, and NaDCL 2, 3 and 4 for further experiments, based on the low variance as indicated by smaller standard errors of the long dsRNA transcript abundance mean values, as well as based on the previous PMRi report of the Arabidopsis dcl triple mutant [15]. Silencing of each NaDCL by the NaDCL 1, 3 and 4, or NaDCL 2, 3 and 4 VIGS construct combinations was confirmed by transcript quantification (Fig S4B- S4E). M. sexta larvae were fed ir-CYP6B46 (30-2) plants infiltrated with these NaDCL VIGS construct combinations. WT as well as ir-CYP6B46 (30-2) plants infiltrated with empty VIGS vector (EV) were used as controls. After feeding on the two combination NaDCL VIGS plants for 14 d, larval midguts showed 50% reductions in CYP6B46 transcripts as compared to those of larvae fed on ir-CYP6B46 (30-2)-EV plants (p#0.05) (Fig. 4C), demon- PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31347