Quantification of viral load

Absolute quantitative two-step RT-qPCR was used to measure the number of genomic copies of TMV. For the standard curve, a 474-pb fragment from positions 1367 to 1699 of the TMV genome of p843pe35TMVr.1 was transcribed using T7 RNA polymerase. The reaction was treated with DNase (Ambion) and RNA amount measured by UV spectrophotometry. RT step was performed using 2 µg of total RNA and 20 pmol of reverse primer qTMV1699R (complete list of primers in Supplementary Table ^S7) at 65 °C for 5 min in a total volume of 7.25 µl. Then, reverse transcriptase 30 U of AMV RT (Promega) and 40 U of RNasin RNA inhibitor (Promega) in a total volume of 20 µl were added to the mix that was incubated for 45 min at 37 °C followed by 5 min at 94 °C. In the second step, 1 µl of the first reaction were mixed with reaction mixture SYBR Green I Master (Roche) and qTMVFor1367 and qTMV1699_R primers (0.5 μM final concentration). Two replicates for each cDNA sample were run using the LightCycler 480 instrument (Roche). For RTqPCR normalization, 25S_universal_F and 25S_universal_R⁷⁰ were used. The PCR protocol consisted of initial denaturalization at 95 °C for 30 s followed by 40 cycles of denaturing at 95 °C for 10 s and primer annealing and extension at 60 °C for 15 s. Standard curves were obtained by linear regression analysis of quantification cycle (C_q) values of three replicates for dilutions (ranging from 10⁹ to 10³ viral copies per μl), with R² of 0.998 and 98% primer efficiency. Number of viral RNA molecules was inferred from this standard curve using Lightcycler software 1.5 (Roche). Additionally, normalization of absolute quantification for each sample was achieved quantifying the 25 S rRNA reference gene⁷⁰.