|Kumar, N.k. krishna|
Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/10/2007
Publication Date: 1/1/2008
Citation: Whitfield, A.E., Kumar, N., Rotenberg, D., Ullman, D.E., Wyman, E.A., Zietlow, C., Willis, D.K., German, T.L. 2008. A soluble form of the tomato spotted wilt virus (TSWV) glycoprotein GN (GN-S) inhibits transmission of TSWV by Frankliniella occidentalis. Phytopathology. 98:45-50. Interpretive Summary: Virus induced gene silencing (VIGS) is a molecular tool with tremendous potential for gene analysis in agronomically important crops. We used the new methodology, real-time RT-PCR, to quantitate the effect of VIGS on a gene required for pigment production in a tobacco host that is permissive for VIGS and in the agronomically important host tomato. During the course of this work, we identified useful tools for the real-time analysis of tobacco and tomato. We have found that VIGS in tomato is highly variable to the point that it has limited usefulness in this crop. Our data shows that this variation is related to the variable replication of the VIGS vector in tomato. This work provides biological targets for improving VIGS in tomato and other crops. We now know that the replication and spread of the viral VIGS vector must be improved to render VIGS a useful molecular tool in agronomically important crops.
Technical Abstract: Tobacco rattle virus (TRV) recombinant viral vectors are commonly used to mediate virus-induced gene silencing (VIGS) of target genes in Nicotiana benthamiana and tomato; however, the efficiency of TRV-based VIGS is more variable in tomato. Our goal was to use real-time RT-PCR to quantify the effect of VIGS on transcript levels of phytoene desaturase (PDS) in leaf tissue of both hosts and monitor the relative abundance of the recombinant TRV vector (rTRV) coat protein (CP) RNA to estimate viral replication. Primer sets designed to amplify the 5' or 3' regions of endogenous PDS transcripts in tomato yielded similar reductions in transcript levels indicating a uniform degradation of target RNA. VIGS in N. benthamiana resulted in complete photo-bleaching of all foliar tissue compared to the chimeric bleaching in tomato. PDS transcript levels were reduced 11 and 7-fold in photobleached leaves of N. benthamiana and tomato, respectively, while sampling tomato leaflets on the basis of age and not phenotypic silencing resulted in only a 17% reduction in PDS coupled with a large leaf-to-leaf variation. There was a significant inverse relationship (R2 = 76%, P = 0.01) between the relative abundance of CP RNA and the amount of PDS transcript in rTRV::tPDS-infected tomato. Our findings in tomato supports the hypothesis that rTRV replication necessitates RNA-silencing and that the silencing inducer acts locally in tissue infected with the virus.