Abstract
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.
Highlights
RNA silencing is an evolutionarily conserved mechanism that regulates gene expression in eukaryotes and meditates defence against invasive nucleic acids such as transposons, transgenes and viruses (Baulcombe, 2004)
A full reduction of Green Fluorescent Protein (GFP) expression could be detected in Tobacco rattle viruses (TRV)-GFP-infected plants kept at 29°C at 21 dpi, whereas TRV-35S-infected plants showed similar GFP expression level to TRV-WT-infected plants (Fig. 2b). These results reveal that virus-induced transcriptional gene silencing (ViTGS) and virus-induced posttranscriptional gene silencing (ViPTGS) act differently at high temperatures
We observed a gap in TRV-GFP-2M primary small interfering RNA (siRNA) alignment at 21 dpi, which was a result of virus-derived primary siRNA (GFP-2M-specific siRNAs) elimination. These results indicate that ViPTGS is associated with large amount of secondary siRNA production, and these secondary siRNAs are sufficient to maintain the ViPTGS in the absence of primary siRNAs
Summary
RNA silencing is an evolutionarily conserved mechanism that regulates gene expression in eukaryotes and meditates defence against invasive nucleic acids such as transposons, transgenes and viruses (Baulcombe, 2004). In the case of RNA viruses, the RISC disables the virus by cleavage of the RNA genome in a manner analogous to PTGS, while DNA viruses are repressed by DNA methylation, analogous to TGS mechanisms (Ghoshal & Sanfacon, 2015; Guo et al, 2019). This innate immune strategy has been applied to develop a method for silencing endogenous genes in plants, known as virus-induced gene silencing (VIGS) (Ruiz et al, 1998). Virus-derived siRNAs (vsiRNAs) that are homologous to the target locus are generated and can knock down expression of the target gene through RNA silencing mechanisms
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