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United States Department of Agriculture

Agricultural Research Service

Title: Real-Time Qrt-Pcr Analysis of Virus-Induced Gene Silencing (Vigs) in Nicotiana Benthamiana and Tomato

Authors
item Willis, David
item Rotenberg, Dorith - UNIV OF WISCONSIN
item German, Thomas - UNIV OF WISCONSIN
item Thompson, Thea - UNIV OF WISCONSIN

Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: January 14, 2005
Publication Date: June 18, 2005
Citation: Rotenberg, D., Thompson, T.S., German, T.L., Willis, D.K. 2005. Real-time qRT-PCR analysis of virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato [abstract]. American Society for Virology Meeting. p. 211-212.

Technical Abstract: Tobacco rattle virus (TRV) recombinant viral vectors are commonly used in virus-induced gene silencing (VIGS) to study gene function in Nicotiana benthamiana and tomato. Our goal was to use real-time qRT-PCR to quantitate the effect of VIGS on transcript levels of phytoene desaturase (PDS), an enzyme involved in carotenoid biosynthesis, in both of these plant hosts. A recombinant vector was constructed by PCR amplifying a 408 bp fragment of the tomato PDS (tPDS) gene and cloning the fragment into the TRV RNA2 (designated RNA2::tPDS) vector pYL156 at a site just 3' to the coat protein (CP) gene. Agrobacterium infection was used to launch expression cassettes containing recombinant TRV (rTRV) into the plants. We followed rTRV replication by measuring the amount of tPDS VIGS RNA and/or CP RNA. We designed primer sets to the 5' and 3' regions of tPDS RNA to examine potential variation in the degradation of different segments of the endogenous target RNA. We evaluated plant gene transcripts ubiquitin, elongation factor-1, and actin as candidates for reference RNAs in real-time qRT-PCR and all three genes were good internal standards in TRV-infected plants. The insert in RNA2::tPDS was stable in both hosts as measured by the ratio of TRV CP RNA to tPDS VIGS RNA. Both 5' and 3' tPDS primer sets yielded similar reductions in RNA levels indicating a relatively uniform degradation of target RNA. While silencing of tPDS in both hosts was relatively similar (60% to 90% reduction in PDS RNA), VIGS in Nicotiana benthamiana resulted in complete photo-bleaching of foliar tissue compared to the patchy, variegated loss of pigment in tomato. Using a primer set specific for the tPDS fragment in RNA2, we were able to compare the level of the tPDS VIGS RNA (essentially the amount of RNA2) to the expression level of the endogenous PDS transcript. Viral symptoms were severe in N. benthamiana which coincided with a remarkably high level of TRV2::tPDS RNA (up to 18,000-fold relative increase). In tomato, viral symptoms were minimal and a maximum 4000-fold relative increase in TRV2::tPDS RNA was detected. Our data indicate that a high level of viral replication is required for efficient VIGS in plants.

Last Modified: 8/1/2014
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