Temperature modulates virus-induced transcriptional gene silencing via secondary small RNAs

Virus-induced gene silencing (VIGS) can be harnessed to sequence-specifically degrade host transcripts and induce heritable epigenetic modifications referred to as virus-induced post-transcriptional gene silencing (ViPTGS) and virus-induced transcriptional gene silencing (ViTGS), respectively. Both ViPTGS and ViTGS enable manipulation of endogenous gene expression without the need for transgenesis. Although VIGS has been widely used in many plant species, it is not always uniform or highly efficient. The efficiency of VIGS is affected by developmental, physiological and environmental factors. Here, we use recombinant Tobacco rattle viruses (TRV) to study the effect of temperature on ViPTGS and ViTGS using GFP as a reporter gene of silencing in N. benthamiana 16c plants. We found that unlike ViPTGS, ViTGS was impaired at high temperature. Using a novel mismatch-small interfering RNA (siRNA) tool, which precisely distinguishes virus-derived (primary) from target-generated (secondary) siRNAs, we demonstrated that the lack of secondary siRNA production/amplification was responsible for inefficient ViTGS at 29°C. Moreover, inefficient ViTGS at 29°C inhibited the transmission of epigenetic gene silencing to the subsequent generations. Our finding contributes to understanding the impact of environmental conditions on primary and secondary siRNA production and may pave the way to design/optimize ViTGS for transgene-free crop improvement.