Characterization of Post-Transcriptionally Suppressed Transgene Expression That Confers Resistance to Tobacco Etch Virus Infection in Tobacco.

Abstract

Tobacco lines expressing transgenes that encode tobacco etch virus (TEV) coat protein (CP) mRNA with or without nonsense codons give rise to TEV-resistant tissues that have reduced levels of TEV CP mRNA while maintaining high levels of transgene transcriptional activity. Two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). Here, we show that at early times in development, immune lines are susceptible to TEV infection and accumulate full-length CP mRNA. Therefore, immune lines also exhibit meiotic resetting, as is seen in the recovery lines, providing molecular evidence for a common mechanism of gene silencing and virus resistance in both cases. We also investigated the characteristics of two sets of low molecular weight RNAs that appear only in silenced tissue. One set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated with polyribosomes; the second set contains the 3[prime] end of the mRNA. Treating silenced leaf tissue with cycloheximide resulted in decreased levels of full-length mRNA and an increase in the levels of the low molecular weight RNAs, supporting a cytoplasmic decay mechanism that does not require ongoing translation. Surprisingly, mRNA from the transgene containing nonsense codons was associated with more ribosomes than expected, possibly resulting from translation from a start codon downstream of the introduced translational stop codons. We present a hypothesis for transgene/viral RNA degradation in which RNA degradation occurs in the cytoplasm while in association with polyribosomes.

Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus.

“…These are the ' immunity ' or ' resistant ' phenotype (R ; no symptoms and no accumulation of virus) and the ' recovery ' phenotype (RC ; systemic symptoms that gradually disappear in newly developed leaves some time after inoculation). Recently Tanzer et al (1997) have shown that R lines inoculated at earlier developmental stages are indeed susceptible to virus infection followed by quick recovery from symptoms, suggesting that both R and RC phenotypes are reversible by meiosis and are manifestations of the same process. Slight differences in the developmental stage could explain our results with line P1-51, which showed mostly asymptomatic, virus-free plants but also had some viruscontaining plants with different degrees of quick recovery.…”

“…When a plant is transformed with a viral sequence, PTGS will suppress accumulation of the transgene mRNA and of any viral RNA with sequence similarity with the transgene, leading to protection (English et al, 1996). PTGS probably occurs by cytoplasmic degradation of the targeted RNAs (Lindbo et al, 1993 ;Sijen et al, 1996 ;Tanzer et al, 1997) and a similar phenomenon has been described recently in nontransgenic, virus-infected plants (Ratcliff et al, 1997).…”

“…The recovery in some P1-51 plants was fast enough to generate symptomless phenotypes scored as R. This ' quick recovery ' in P1-51 was clearly delayed when plants were inoculated with the heterologous strain TVMV-S indicating that, in addition to the developmental stage of the plant, sequence identity with the incoming virus can modulate the speed of the recovery process. Tanzer et al (1997) have proposed that the primary difference between R and RC lines would reside in the need for virus infection to activate silencing. In this model, R lines are associated with silencing of the transgene prior to virus infection.…”

Cosuppression of Eukaryotic Release Factor 1-1 in Arabidopsis Affects Cell Elongation and Radial Cell Division

“…A similar degradation smear below the full-length transcript on a northern has been reported for transgenic N. benthamiana plants in which the LUCIFERASE gene had been silenced (Kjemtrup et al, 1998). In addition, the occurrence of truncated, polyadenylated, and nonpolyadenylated RNAs corresponding to 5# and 3# regions of posttranscriptionally silenced genes has been previously described (Smith et al, 1990;Goodwin et al, 1996;Lee et al, 1997;Metzlaff et al, 1997;Tanzer et al, 1997;Han and Grierson, 2002), suggesting that transgene degradation is gene specific.…”

Plant Development from the Cellular Perspective: Integrating the Signals

“…Among the possibilities he mentioned are the control of vira1 infections, the generation of hybrid vigor, and the tissue-specific regulation of gene families. A number of recent publications (Que et al, 1997;Ratcliff et al, 1997;Tanzer et al, 1997; have provided further insights into both the mechanisms and functions of gene silencing in plants, including the tantalizing suggestion that signals triggering cosuppression can move .…”