Virus-Induced Gene Silencing as a Reverse Genetics Tool to Study Gene Function

Bernacki, Steven; Karimi, Mansour; Hilson, Pierre; Robertson, Niki

doi:10.1007/978-1-60761-765-5_3

Cited by 10 publications

(3 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Virus-induced gene silencing (VIGS) is a particularly 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 ). VIGS is based on the plant’s endogenous RNA defense response that recognizes the accumulation of foreign double-stranded RNAs (dsRNA) and targets these sequences for degradation ( Ding, 2000 ).…”

Section: Introductionmentioning

confidence: 99%

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 99%

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature

et al. 2018

View full text Add to dashboard Cite

show abstract

“…The high-throughput VIGS process involves cloning libraries of short gene fragments, such as ESTs, directly into a VIGS vector, inoculating plants, and then identifying phenotypes of interest (Bernacki et al , 2010; Lu et al , 2012). Moreover, this work studied the cooperative action of different classes of MADS-box TFs by co-silencing with separate VIGS vectors.…”

Section: Introductionmentioning

confidence: 99%

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

Hsieh

Pan

Lai

et al. 2013

Journal of Experimental Botany

View full text Add to dashboard Cite

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

show abstract

“…Reverse genetics collectively refers to approaches aiming to interrogate the gene functions via analysis of the phenotypes caused by specifically engineered or mutated gene sequences 38 – 40 . The reverse genetic screens are most informative when the analysed phenotypes are easy to discern, quantitatively measured and highly penetrant.…”

Section: Discussionmentioning

confidence: 99%

Phenotype-driven identification of epithelial signalling clusters

Marques

Peltola

Kaski

et al. 2018

Sci Rep

View full text Add to dashboard Cite

In metazoans, epithelial architecture provides a context that dynamically modulates most if not all epithelial cell responses to intrinsic and extrinsic signals, including growth or survival signalling and transforming oncogene action. Three-dimensional (3D) epithelial culture systems provide tractable models to interrogate the function of human genetic determinants in establishment of context-dependency. We performed an arrayed genetic shRNA screen in mammary epithelial 3D cultures to identify new determinants of epithelial architecture, finding that the key phenotype impacting shRNAs altered not only the data population average but even more noticeably the population distribution. The broad distributions were attributable to sporadic gene silencing actions by shRNA in unselected populations. We employed Maximum Mean Discrepancy concept to capture similar population distribution patterns and demonstrate here the feasibility of the test in identifying an impact of shRNA in populations of 3D structures. Integration of the clustered morphometric data with protein-protein interactions data enabled hypothesis generation of novel biological pathways underlying similar 3D phenotype alterations. The results present a new strategy for 3D phenotype-driven pathway analysis, which is expected to accelerate discovery of context-dependent gene functions in epithelial biology and tumorigenesis.

show abstract

Virus-Induced Gene Silencing as a Reverse Genetics Tool to Study Gene Function

Cited by 10 publications

References 31 publications

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

Phenotype-driven identification of epithelial signalling clusters

Contact Info

Product

Resources

About