RNA extraction and RT-qPCR.

Total RNA was extracted from 100-mg samples of N. benthamiana leaf tissue using the Qiagen RNeasy plant minikit, and residual DNA was digested using DNase I (Promega). RNA extraction from purified virions was performed using phenol-chloroform, after treatment with micrococcal nuclease (New England Biolabs) to digest extraneous, nonencapsidated RNA, similarly to other studies (21, 26). The RNA quantity and purity were assessed using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific), and 1 μg of RNA was used in a reverse transcription reaction with random hexamers (Promega) and Superscript III reverse transcriptase (Thermo Fisher Scientific). The qPCR assays were performed in the OneStepPlus real-time qPCR system (Applied Biosystems) using the Power SYBR green PCR master mix (Applied Biosystems) and the relative standard curve quantitation method. All oligonucleotide primers were designed by Primer-BLAST (39) and are available upon request; all reagents were used according to the manufacturer’s instructions. Control reactions using RNA prior to reverse transcription were also included to exclude the possibility that DNA templates were still present.