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

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.

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Stability of Barley stripe mosaic virus–Induced Gene Silencing in Barley

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-11-1323 ◽

2007 ◽

Vol 20 (11) ◽

pp. 1323-1331 ◽

Cited By ~ 89

Author(s):

Marianne Bruun-Rasmussen ◽

Christian Toft Madsen ◽

Stine Jessing ◽

Merete Albrechtsen

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Barley Stripe Mosaic Virus ◽

Mrna Levels ◽

Virus Induced Gene Silencing ◽

Qrt Pcr ◽

Transient Nature ◽

Polymerase Chain ◽

Insert Length ◽

Pds Gene

Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting photobleaching in infected barley plants was used as a reporter for silencing. In addition, downregulation of PDS mRNA was measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Using fragments of PDS ranging from 128 to 584 nucleotides in BSMV, we observed that insert length influenced stability but not efficiency of VIGS. Silencing was transient in most cases; however, the decrease in PDS mRNA levels measured by qRT-PCR began earlier and lasted longer than the photobleaching. Occasionally, silencing persisted and could be transmitted through seed as well as via mechanical inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector.

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A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing

Plasmid ◽

10.1016/j.plasmid.2009.04.001 ◽

2009 ◽

Vol 62 (1) ◽

pp. 50-55 ◽

Cited By ~ 1

Author(s):

James R. Gilbert ◽

Christopher S. Adams ◽

Irving M. Shapiro ◽

Noreen J. Hickok

Keyword(s):

Gene Silencing ◽

Expression System ◽

Short Hairpin Rna ◽

Hairpin Rna ◽

Shrna Expression ◽

Short Hairpin

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Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology

Frontiers in Plant Science ◽

10.3389/fpls.2021.610283 ◽

2021 ◽

Vol 12 ◽

Author(s):

Neeti Sanan-Mishra ◽

A. Abdul Kader Jailani ◽

Bikash Mandal ◽

Sunil K. Mukherjee

Keyword(s):

Gene Silencing ◽

Small Rnas ◽

Rna Silencing ◽

Viral Infections ◽

Natural Resistance ◽

Virus Induced Gene Silencing ◽

Systemic Spread ◽

Secondary Sirnas ◽

Silencing Pathways ◽

Virus Interactions

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and trans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant–virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

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