Quantification of TYLCV Infection Dynamics

TYLCV infected and control plants were monitored along 21 days. The second most recently expanded leaf from the apex was harvested at 2, 7, 14 and 21 days-post infection (dpi). The presence of viral DNA was analysed in each test plant at 21 dpi by hybridization of tissue blots^{64 (link)}. For each infection method, time point and replica, the leaf tissue from 6 infected plants was pooled and used in downstream analysis. A total of three biological replicates were processed per condition and time point.
Total DNA was isolated with the DNeasy Plant Mini Kit (Qiagen). Total RNA was isolated using the Trizol reagent (Ambion), and 3 μg were set aside for construction and sequencing of small RNAs libraries. Total RNA was treated with RNase free Turbo DNaseI (Ambion) according to the manufacturer’s guidelines and cleaned up by a phenol:chloroform treatment.
Absolute quantification of the virion-sense (VS) and the complementary-sense (CS) strands was performed following the protocol described by⁵² with the following modifications: 15 ng of total DNA were used for the VS and CS strand synthesis step and 2 μl of a 1:2 dilution of the purified DNA, were used as the template for the qPCR. Total viral DNA was quantified by standard qPCR using 3 ng of total DNA.