Abstract
Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.
Highlights
Virus-induced gene silencing (VIGS) is a powerful tool for studying plant functional genomics, especially for those organisms where genetic transformation is difficult (Burch-Smith et al, 2004; Robertson, 2004; Bernacki et al, 2010; Barciszewska-Pacak et al, 2016)
We developed a Chinese wheat mosaic virus (CWMV)-based VIGS vector that silenced the phytoene desaturase (PDS) gene at 17◦C in both monocot and dicot (N. benthamiana) hosts more effectively than a vector based on Barley stripe mosaic virus (BSMV), Foxtail mosaic virus (FoMV), or Tobacco rattle virus (TRV)
We demonstrate that the CWMV vector is effective in silencing endogenous miRNAs using short tandem target mimic (STTM)
Summary
Virus-induced gene silencing (VIGS) is a powerful tool for studying plant functional genomics, especially for those organisms where genetic transformation is difficult (Burch-Smith et al, 2004; Robertson, 2004; Bernacki et al, 2010; Barciszewska-Pacak et al, 2016). Many plant viruses multiply in their hosts using a dsRNA intermediate, which is recognized as foreign dsRNA by the host endogenous RNA defense systems. This dsRNA, including the inserted target gene in VIGS studies, is processed into the RNA interference pathway to degrade the complementary sequence (Denli and Hannon, 2003). BSMV-based VIGS or TRV-based VIGS was widely applied in study gene function of wheat as well in many dicots, respectively. It is important to develop a convenient and efficient VIGS vector suitable for gene silencing in both monocots and dicots, especially important for wheat
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