Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.

Plant Methods
Enwu Liu, Jonathan E Page

Abstract

Virus-induced gene silencing (VIGS) has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS) gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV). NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A) or poly(G) homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A) tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmo...Continue Reading

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Jul 12, 2008·Molecular Plant-microbe Interactions : MPMI·Michael M GoodinSteven A Lommel
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Datasets Mentioned

BETA
EU165355
EU165356

Methods Mentioned

BETA
PCR
EST sequencing
cDNA library

Software Mentioned

VIGS

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