DNA Quantification by qPCR

DNA quantification (10 biological replicates per accession and treatment, Supplementary Figure S1) was performed as duplicated qPCR in 384-well optical plates using the LightCycler FastStart DNA Master Plus SYBRGreen I kit (Roche) in a LightCycler 480 (Roche) thermocycler according to the manufacturer’s instructions. Specific primers designed for the quantification of CaMV genome (Ca4443-F: 5′-GACCTAAAAGTCATCAAGCCCA-3′ and Ca4557-R: 5′-TAGCTTTGTAGTTGACTACCATACG-3′) and A. thaliana ubiquitin-conjugating enzyme 21 gene (UBC21; UBC21_At_F: 5′-TGCAACCTCCTCAAGTTCGA-3′ and UBC21_At_R: 5′-GCAGGACTCCAAGCATTCTT-3′) were used at a final concentration of 0.3 μM. All qPCR reactions were performed with 40 cycles (95°C for 15 s, 62°C for 15 s and 72°C for 15 s) after an initial step at 95°C for 10 min. The qPCR data were analyzed with the LinReg PCR program to account for the efficiency of every single PCR reactions (Ruijter et al., 2009). The absolute initial viral concentration in A. thaliana plants, expressed in arbitrary fluorescence units (N₀ CaMV) was divided by that of A. thaliana UBC21 gene (N₀ UBC21; Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"DQ027035","term_id":"66354451","term_text":"DQ027035"}}DQ027035), in order to normalize the amount of plant material analyzed in all samples.