Development of a Viral RdRp-Assisted Gene Silencing System and Its Application in the Identification of Host Factors of Plant (+)RNA Virus.

Wang Zhang,Liqun Wang,Haijian Zhi,Jinlong Yin,Kai Xu,Yanglin Qiu,Lingyun Zhou

doi:10.3389/fmicb.2021.682921

Wang Zhang, Liqun Wang + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2021.682921

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Abstract

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions.

Highlights

Gene silencing is a cellular mechanism that acts on both transcriptional and posttranscriptional levels to regulate gene expression
To circumvent the use of infectious virus but still exploit the ability of viral RNA-dependent RNA polymerase (RdRp) in amplifying the RNA template, we have developed a two-vector-based system to initiate the synthesis of doublestranded RNA (dsRNA) in vivo for the induction of gene silencing
The positive-strand initiation promoter supported 4∼5-fold higher complementary RNA synthesis than the promoter for the negative strand. These results demonstrated that the higher silencing efficiency in the “pTNV(+)-phytoene desaturase (PDS) + p82C” combination is due to more active promoter activity that leads to more dsRNA production

Summary

Introduction

Gene silencing is a cellular mechanism that acts on both transcriptional and posttranscriptional levels to regulate gene expression. DsRNA in the form of an intron-spliced hairpin RNA (ihpRNA) was demonstrated to be effective in inducing the posttranscriptional gene silencing (PTGS), comparing with the single-stranded sense or antisense RNA fragments or the hairpin RNA without intron as the spacer (Smith et al, 2000; Wesley et al, 2001). A model system based on transient expression of ihpRNA in Nicotiana benthamiana leaves showed that the transcripts level of targeted ectopically expressed β-glucuronidase or green fluorescence protein-coding gene only reduced 76% or 64% (Yan et al, 2012). The remaining transcripts of target genes could still be translated and contribute to the phenotype, limiting the use of ihpRNA-induced PTGS in genetics studies

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