Total RNA was extracted from leaf tissues of maize, N. benthaniana, 16C, and Cucumis melo plants using the Transzol reagent (TransGen Biotech, Beijing, China) and then treated with a gDNA wipe enzyme (Vazyme, Nanjing, China) to eliminate the genomic DNA. The first-strand cDNA for RT-PCR was synthesized from 500 ng total RNA by HiScript® II Q RT SuperMix kit (Vazyme, Nanjing, China) following the manufacturer’s instructions.
The qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a PCR machine (LC96, Roche, Basel, Switzerland). Gene-specific primers (Supplemental Table S1 XM_008647047 ), N. benthamianaactin gene (GenBank accession: AY179605 ), and Cucumis meloEF1α gene (GenBank accession: XM_008459007 ). The accumulation levels of these genes are stable during viral infection and are therefore used as internal controls for qRT-PCR (Gao et al., 2012 (link); Sun et al., 2014 (link); Chen et al., 2017 (link)). Each qRT-PCR was performed with three biological replicates.
The qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a PCR machine (LC96, Roche, Basel, Switzerland). Gene-specific primers (
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