Figure S1. Elements in the promoter sequence ofABCG5/PDR5. The ABCG5/PDR5 transcription promoter region contains a stop codon at position -18 (bold) in frame with the translation initiation codon (bold and underlined), and a T/G-box at position -456 bp (italics and bold).

Figure S2. Phylogenetic analysis of PDRtransporters from different species. PDR amino acid sequences fromNt (N. tabacum), Np (N. plumbaginifolia), At(Arabidopsis thaliana), Os (Oryza sativa), Gm(Glycine max), Sp (Spirodela polyrrhiza) werecompared using the CLUSTALW program and an unrootedneighbor-joining tree generated( novel ABCG5/PDR5 isoforms of N. tabacum and N.plumbaginifolia are shown in blue. Bootstrap values for the branches are shown. The distance scale represents the evolutionary distance expressed as the number of substitutions per amino acid.

Figure S3. Sequence motifs in ABCG5/PDR5. ABCG5/PDR5, here exemplified by NtPDR5a, contains the typical PDR motifs (gray), the Walker A and Walker B sequence motifs and the ABC signature motifs (highlighted).

Figure S4. Immunogenic sequence of the ABCG5/PDR5 antibody. A 61 amino acid sequence in a unique region of ABCG5/PDR5 (underlined) was chosen for immunizing rabbits and obtaining an isoform specific antibody.

Figure S5. Subcellular localization of PDR5 inN. tabacum. (a-f) Localization of the GFP-PDR5 (a) andPDR5-GFP (d) fusion proteins and the plasma membrane marker FM4-64(b and e), and the merged images (c and f) in a single planeconfocal image. Bars = 10 μm.

Figure S6. Illustration of wounding of N.tabacum plants. Wounding was either realized using forceps pressure or a fabric pattern to perforate the leaf tissue. A control sample was always taken from a non-injured area on the same leaf.

Figure S7. Silencing of ABCG5/PDR5 doesnot affect PDR1 expression. Microsomal fractions of hydroponicallygrown N. tabacum roots were isolated and subjected toimmunoblotting with an ABCG5/PDR5-specific antibody, aPDR1-specific antibody and a general anti-H+-ATPaseantibody as a loading control.

Figure S8. Silencing of NpPDR1 in N.tabacum. NpPDR1 expression was silenced by RNAinterference as previously described in Stukkens et al. (2005).Microsomal fractions of hydroponically grown N. tabacumroots were isolated and subjected to immunoblotting with aNpPDR1-specific antibody and a general anti-H+-ATPaseantibody as a loading control. Two independet lines,Nppdr1-1 and Nppdr1-2, were analyzed induplicates.

Figure S9. HPLC chromatograph of N.tabacum leaf extracts and selected peak quantification.Methanolic leaf extracts from wild type, pdr5-1, andpdr5-2 were separated and quantified by HPLC-UV.Concentrations of Nicotine (a), Chlorogenic Acid (b), DicaffeoylSpermidine (c), or Rutin (d) in wild type, pdr5-1, andpdr5-2, that were wounded and treated with oral secretions.Error bars represent standard deviation; no significant differencewas found between the different extracts.

Figure S10. Soluble sugars analysis of N.tabacum leaf extracts. Concentrations of the soluble sugarsGlucose (a), Fructose (b), and Sucrose (c) in leaf extracts fromwild type, pdr5-1, and pdr5-2 plants were determined using an ethanolic extraction and a coupled enzymatic assay; concentrations shown were measured at absorbance 340 nm. Error bars represent standard error; no significant difference in any quantified sugar was found between the extracts.

Figure S11. Primers used for the amplificationof amiRNA constructs. Two artificial micro RNA (amiRNA) constructsdirected against regions in ABCG5/PDR5 were obtained asindicated in Methods and introduced in N. tabacum by A.tumefaciens mediated transformation.

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