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Figure S1. Biosynthetic pathways leading to the formation of benzylisoquinoline alkaloids identified in extracts of opium poppy latex. Corresponding cDNAs have been isolated for enzymes shown in blue. Duplicate arrows represent multiple enzymatic steps.

Figure S2. Regions of SalSyn, SalR, SalAT, T6ODM, COR and CODM cDNAs used to construct pTRV2 vectors for virus-induced gene silencing are shown in red. The scale is in base pairs (bp). The white bars and the black lines represent open reading frames and untranslated regions, respectively. The nucleotide sequences of primers used to amplify each gene fragment are provided in Table S3.

Figure S3. Reverse transcription (RT)-PCR analysis of opium poppy plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct. Amplicons of 613 and 216 base pairs were obtained using primers specific to tobacco rattle virus-coat protein (TRV-CP) and opium poppy glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers, respectively. The occurrence of TRV-CP transcripts confirms the presence of the tobacco rattle virus. GAPDH amplicons were used as a positive PCR control.

Figure S4. Real-time quantitative (RT-q)PCR using primers specific to known morphinan alkaloid biosynthetic genes in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black bars) compared with empty pTRV2 vector controls (white bars). Suppression of targeted gene transcript levels compared with controls is shown in Figure 2. Each bar represents the mean ± standard deviation of 3 technical replicates on each of 3 biological replicates from 6 individual plants. No significant difference in the transcript level of any peripheral biosynthetic gene was detected using a Tukey-Kramer pair-wise comparison (α  =  0.05) in plants that showed significant silencing of targeted transcripts compared with controls.

Figure S5. Correlations between targeted gene transcript and reticuline levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S6. Correlations between targeted gene transcript and salutaridine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S7. Correlations between targeted gene transcript and thebaine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S8. Correlations between targeted gene transcript and codeine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S9. Correlations between targeted gene transcript and morphine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S10. Correlations between targeted gene transcript and noscapine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Figure S11. Correlations between targeted gene transcript and papaverine levels in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 vector construct (black circles) compared with empty pTRV2 vector controls (white circles). a, SalSyn; b, SalR; c, SalAT; d, T6ODM; e, COR; f, CODM.

Table S1. Oligonucleotide primers used to construct pTRV2 vectors for virus-induced gene silencing and for RT-qPCR analysis.

Table S2. Chromatographic and mass spectral data used for the identification and quantification of benzylisoquinoline alkaloids by LC-MS.

Table S3. Relative abundance of benzylisoquinoline alkaloids identified by LC-MS in plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 construct. Values indicate the percentage of individual alkaloids compared with the total of all 22 identified alkaloids and represent the mean ± standard deviation of 6 individual plants.

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