Total RNA Extraction, RT-PCR, and Real-Time PCR

Plant total RNA was extracted using TRIzol Reagent (Life Technologies, USA) and treated using the TURBO DNA-free™ Kit (Invitrogen, USA) to eliminate genomic DNA contamination before reverse transcription. First-strand cDNA was synthesized from 0.5 μg total RNA by M-MLV reverse transcriptase (ThermoFisher Scientific, USA) using a mixture of oligo (dT₁₈) and random primers. Cloning of BMV-RNA3 insert fragments and analysis of BMV insert stability were performed by PCR using Phusion® High-Fidelity DNA Polymerase (NEB, USA) and Taq DNA Polymerase (NEB, USA), respectively, following the manufacturer's instructions. Primer pair P4-F/P4-R flanking the foreign gene insert site in pC13/F3CP5 was used to determine insert stability in both N. benthamiana and wheat through RT-PCR analysis. Gene expression analyses were evaluated by real-time PCR (qPCR) using gene-specific primers (not amplifying the insert) and the Power SYBR Green PCR Master Mix kit (Applied Biosystems, USA) on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, USA). The wheat translation elongation factor subunit EF1α was used as an internal control for gene expression analyses (Paolacci et al., 2009). All primers used for RT-PCR or qPCR in this study were listed in Supplementary Table 3.