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Figure S1. Detailed cDNA and protein deduced sequence description of the fusion HSVd-GFP. The partial GFP sequence is underlined. The primer VP/atg-HSVd carrying the insertion ATG (in bold) is also shown. Figure S2. A) RT-PCR amplification of GFP and HSVd-GFP mRNAs. The total RNAs were extracted according the TRIZOL method (described in text), starting from leaves (0,15 g) of GFP-wt and HSVd-GFP agroinfiltrated and no-infiltrated N. benthamiana plants. RT-PCR analysis of the transgenic plants was performed as previously described (G?mez and Pall?s, 2006). The RT-PCR products were analyzed in 1% agarose gels. In (A) electrophoretic analysis of both amplified cDNA fragments , HSVd-GFP ≅ 1000 bp (lane 1), GFP-wt ≅ 700 bp (lane 2). Lane 3, negative control. The amplified cDNAs were gel extracted, rechecked by electrophoresis in 1% agarose and sequenced. The sequence analysis shows that GFP-wt and HSVd-GFP cDNA amplified from their respective mRNAs expressed in agroinfiltrated plants are coincident in size with the sequences of the original clones. B) The HSVd-GFP protein shows an anomalous electrophoretic behavior. Total proteins were extracted (as described in the text) from leaves (0,15 g) of GFP-wt and HSVd-GFP agroinfiltrated and no-infiltrated N. benthamiana plants, electrophoresed in 15% SDS-PAGE and analyzed by western blot assays (as described in the text). In this more resolutive SDS-PAGE, differential electrophoretic mobility of both proteins (HSVd-GFP, lane 1 and wt-GFP lane 3) can be observed. However the migration pattern of HSVd-GFP protein is different to the expected for a polypeptide of approximately 36 kDa. Figure S3. The capacity to suppress the HSVd-GFP reporter expression is maintained in N. benthamiana, HSVd-transgenic plants. A) The HSVd-GFP expression in 120 day-old N. benthamiana HSVd-transgenic plants was monitored as described above. The green fluoresce was not observed in the HSVd-transgenic N. benthamiana plants agroinfiltrated with the reporter (panel 4). Normal green fluorescence was observed in HSVd-transgenic plants GFP-infiltrated (panel 2) and in the untransformed N. benthamiana HSVd-GFP (panel 3) and GFP (panel 1) infiltrated controls. The basal red fluorescence level was used as auto fluorescence controls. B) Western blot assays (panel II) showing the HSVd-GFP suppression in proteins extracts obtained from agroinfiltrated 120 day-old HSVd-transgenic plants. The HSVd-GFP was no detected in the N. benthamiana transgenic lines agroinfiltrated with the reporter, even if more concentrated samples were analyzed (lines 5 to 7; lanes 5 and 6 are 4x and 2x more concentrated than line 7). GFP expression (marked with arrows) was detected in HSVd-transgenic plants GFP infiltrated (lanes 1 and 2) and in the untransformed N. benthamiana HSVd-GFP (lanes 3 and 4). A duplicate 10% SDS-PAGE was silver stained and used as load control (panel I).

FilenameFormatSizeDescription
TPJ_3203_sm_FigS1.tif4402KSupporting info item
TPJ_3203_sm_FigS2.tif704KSupporting info item
TPJ_3203_sm_FigS3.tif4565KSupporting info item

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