Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs

Abstract

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.

How and Why Do Plants Inactivate Homologous (Trans)genes?

“…Contrary to this expectation, however, are two recent reports claiming that transcriptional inactivation is not necessarily an inevitable consequence of DNA methylation: posttranscriptional silencing of transgenes was correlated with increased methylation at some sites, i.e. transgenes that had become methylated continued to be transcribed (Ingelbrecht et al, 1994;Smith et al, 1994). Although these results appear at first glance to contradict the current view that DNA methylation represses transcription, it is possible that methylation had not yet achieved a density sufficient for complete inactivation of the 35s promoter (which was used in both studies).…”

“…Plants that transcribed the transgene at high levels accumulated only low levels of the untranslatable sense coat protein transcript and were resistant to potato virus Y, again implying the action of an RNA turnover mechanism induced by elevatetl coat protein RNA levels (Smith et al, 1994). The transcribed coat protein transgenes were methylated at the few sites examined.…”

“…Additional ideas about silencing mechanisms and different points at which it can occur in the pathway from DNA to protein have bem discussed by Flavell (1994). Lindbo et al (1993) and Smith et al (1994) have elaborated on the connection between RhIA virus resistance and silencing of nuclear transgenes. An inverted repeat is shown in A, but direct repeats or more than tvio copies can also pair (Dorer and Henikoff, 1994).…”

“…However, B and C might be connected if elevated RNA levels lead to both RhIA turnover in the cytoplasm (C) and de novo methylation of nuclear genes (B). Whether such methylation eventually leads to complete transcriptional inactivation is still not known; a modest degree of apparent RNA-induced methylation does not necessarily result in complete transcriptional inactivation (Ingelbrecht et al, 1994;Smith et al, 1994). Additional ideas about silencing mechanisms and different points at which it can occur in the pathway from DNA to protein have bem discussed by Flavell (1994).…”

“…Although these results appear at first glance to contradict the current view that DNA methylation represses transcription, it is possible that methylation had not yet achieved a density sufficient for complete inactivation of the 35s promoter (which was used in both studies). Indeed, Smith et al (1994) proposed that the apparent RNA-induced transgene methylation that they observed represented "an attempt to reduce transgene transcription." Presumably, the transgene would eventually cease to be transcribed when a certain leve1 of methylation was attained.…”