Fig. 4

RDR activity, but not MtRDR1 transcript accumulation, is induced by SA treatment in MtRDR1-transgenic N. benthamiana plants. Semi-quantitative RT-PCR analysis of PR1 transcript accumulation in leaves of (a) transgenic (empty vector) control and (b) MtRDR1-transgenic plants infiltrated with a control solution of 0.05 % (v/v) ethanol or a solution of 1 mM SA in 0.05 % (v/v) ethanol. Infiltrated leaf tissue samples were harvested for RNA extraction at 72 h post-infiltration. PR1 transcript accumulation levels were determined by RT-PCR after 40 cycles of PCR and compared relative to the accumulation levels of the elongation factor 1 alpha (EF1α) transcript. Increased PR1 accumulation confirmed that SA was taken up by the tissues and was effective in inducing transcriptional changes. c Semi-quantitative RT-PCR analysis showed that there was little difference in MtRDR1 transcript accumulation in MtRDR1-transgenic plants infiltrated with control solution or 1 mM SA. NbRDR1m transcript accumulation was up-regulated in both transgenic control and MtRDR1-transgenic N. benthamiana plants after SA treatment, although the NbRDR1m protein itself is non-functional. MtRDR1 and NbRDR1m transcript accumulation levels after 27 and 35 cycles, respectively, were compared relative to the accumulation levels of EF1α. Infiltrated tissue samples were harvested at 72 h post-infiltration. d RT-qPCR analysis of MtRDR1 transcript levels in leaves of empty vector control and MtRDR1-transgenic plants infiltrated with water control or 1 mM SA solution. MtRDR1 was not detected in empty vector control plants. Mean values for relative MtRDR1 levels (based on duplicate technical replicate values; 100 = mean value for the transcript level in untreated MtRDR1 plants) obtained from three plants (one plant = one independent sample) have been given for each treatment group. Error bars represent standard errors of the mean for the three samples. Relative transcript levels of MtRDR1 were calculated using the 2^-ΔΔC(t) method [59] using EF1α as an internal reference. e Enhancement of RDR activity by SA in MtRDR1-transgenic plants. Leaves from tobacco (N. tabacum, included as a positive control) and transgenic empty vector control and MtRDR1-transgenic N. benthamiana plants were infiltrated with water (-) containing 0.05 % (v/v) ethanol or 2.5 mM SA in 0.05 % (v/v) ethanol (+) and harvested after 48 h for preparation of RDR1-enriched extracts. The proxy for RDR1 activity was the incorporation of α-[³²P] CTP into nascent RNA analysed by liquid scintillation counting of radioactivity incorporated (counts per minute) into trichloroacetic acid-precipitable material. Error bars are standard errors for the mean for three technical replicates (RDR assays) per sample