PCR primers. Sequence alignments of M. sexta CYP cDNA fragments that were selected for PMRi experiments, with the homologous sequences of respective allied genes that were tested for off-target co-silencing effects (see Fig. 1A) (A) Alignment of the selected CYP6B46 cDNA fragment with the homologous fragment from the CYP6B45 cDNA. (B) Alignment of selected cDNA fragments of CYP4M1 and CYP4M3 with each other and with the homologous cDNA fragment from CYP4M2. (C) RT-PCR analysis showing that the primers used for the SYBR-Green qRT-PCR produced single amplicons (resolved on 2% agarose gel) with M. sexta cDNA and did not produce amplicons with N. attenuata leaf cDNA, demonstrating that the primers were insect cDNA specific. A 100 bp ladder was used as a size marker. (TIF) Figure S2 Map of plant stable transformation vector and detection of a single transgene insertion in the transformed lines. (A) A map of pSOL8 vector used for Agrobacterium tumefaciens mediated plant transformation harboring the inverted repeat (separated by the pdk i3 intron) of a 312 bp fragment of M. sexta’s CYP6B46 cDNA. (B) Southern hybridiza- tion after HindIII digested genomic DNA, showing the presence of a single insertion of transgene in both the independently transformed (30-2 and 416-3) N. attenuata lines; WT control shows absence of transgene insertion. 1 kb DNA ladder was used as a size marker. (TIF) Figure S3 Map of viral dsRNA-producing system (VDPS) vector. A map of tobacco rattle virus (TRV) based plant (transient) transformation vector pTV. A $300 bp stretch from M. sexta’s CYP6B46/CYP4M1/CYP4M3 cDNA was cloned in an antisense orientation into pTV for VDPS. N. attenuata (WT) plants were transiently transformed with this recombinant vector by Agro-infiltration and M. sexta larvae were fed on the leaves of inoculated plants to silence the expression of CYP6B46/ CYP4M1/CYP4M3 genes, respectively, in their midguts. (TIF) Figure S4 Schematic representation of cDNA regions used for MsCYP6B46 transcript profiling in leaves and References 1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. 2. Huvenne H, Smagghe G (2010) Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review. Journal of Insect Physiology 56: 227–235. 3. Hutvagner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Current Opinion in Genetics & Development 12: 225–232. 4. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. 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