Plant Mediated RNAi Silencing Manduca Sexta Genes Figure 4. PMRi efficiency is increased after silencing N. attenuata’s dicer-like (DCL) genes. (A) Schematic representation of the silencing of N. attenuata’s four DCLs by virus induced gene silencing (VIGS), in ir-CYP6B46 (30-2) stably-transformed plants. ir-CYP (30-2) plants were Agro- infiltrated with pTVDCL harboring cultures (individually, and in all combinations of DCL1, DCL2, DCL3 and DCL4); pTV (EV) was used as a control. (B) Abundance, relative to NaActin, of a 102 bp region of the 59 end of the 312 bp MsCYP6B46 fragment that the ir-CYP (30-2) N. attenuata plants were harboring in their genome. The plants had been previously Agro-infiltrated with EV or all combinations of vectors designed to silence the expression of the four NaDCLs. (C) Transcript abundance of CYP6B46 (relative to ubiquitin) in the midguts of 4th instar M. sexta larvae, when fed N. attenuata leaves containing no MsCYP6B46 dsRNA (WT+EV), small (diced) MsCYP6B46 dsRNA (ir-CYP6B46+EV) and on leaves of plants containing higher concentration of longer (102 bp detected by qPCR) MsCYP6B46 dsRNA fragments (ir-CYP6B46+ DCL134 and ir-CYP6B46+ DCL234). See Fig. S4A and Table S1 for the design of the primers used in the transcript quantification. Bars labeled with different letters indicate significant differences as determined by one way ANOVAs (p#0.05). doi:10.1371/journal.pone.0031347.g004 family. Such specificity and reproducibility was important to demonstrate, as the RNAi in Lepidoptera has recently been suggested to be dependent on the insect, gene, gene function, organ of expression and mode of delivery [2,24]. More importantly for our aims, this work demonstrates that the M. sexta- N. attenuata ecological model was amenable to PMRi. PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31347