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United States Department of Agriculture

Agricultural Research Service

Title: Real-Time Qrt-Pcr Analysis of Vigs in Nicotiana Benthamiana and Tomato

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 11, 2004
Publication Date: September 14, 2004
Citation: Willis, D.K., Thompson, T.S., Rotenberg, D., German, T.L. 2004. Real-time qrt-PCR analysis of VIGS in Nicotiana benthamiana and tomato. p. 1-12.

Technical Abstract: The difference in efficiencies of virus induced gene silencing (VIGS) between Nicotiana benthamiana and tomato was quantitated using Real-Time qrt-PCR. N. benthamiana is a very good host for VIGS while tomato gives a variable VIGS reaction. The PDS gene encodes phytoene desaturase and reduction in its expression results in an easily detectable phenotype, the loss of leaf pigment and the appearance of white leaves. We used tobacco rattle virus (TRV) containing a 350 bp fragment of the tomato PDS as the VIGS vector. We measured the PDS gene transcript using three different primer sets specific for the 5' and 3' ends of the transcript and for fragment of the transcript contained in the VIGS vector. We also used a primer specific for the TRV coat protein to measure the amount of viral expression. We investigated three transcripts, ubiquitin (ubi3), elongation factor 1 (EF1), and actin (act41) for use as internal standards. With the exception of a primer set specific for the 5' end of the PDS transcript, all primers gave equivalent efficiencies on both N. benthamiana and tomato. Ubiquitin and EF-1 proved to be suitable internal standards in both plant species, while actin worked well in N. benthamiana but was variable in tomato. Our data shows that TRV based VIGS quantitatively reduced PDS transcript levels in both plant species. The PDS transcript was reduced from 75 to 80% in tomato and 80 to 99% in N. benthamiana. By determining the ratio of the PDS silencing transcript to the TRV coat protein transcript, we established that there was no appreciable loss of the silencing transcript from the TRV vector in either plant host. The most striking difference between the two plant hosts was the remarkable expression of the PDS VIGS transcript from TRV in N. benthamiana. This transcript increased to a maximum of 18,000-fold higher than the endogenous PDS transcription from the plant chromosome. In tomato, this ratio rarely exceeded 1,000-fold. Taken together, our data suggests that the increased efficiency of VIGS in N. benthamiana is most likely due to the dramatic increase of viral transcripts in this plant host.

Last Modified: 12/19/2014
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