Abstract
The 35S promoter with a duplicated enhancer (frequently referred to as 2X35S) is a strong dicotyledonous plant-specific promoter commonly used in generating transgenic plants to enable high-level expression of genes of interest. It is also used to drive the initiation of RNA virus replication from viral cDNA, with the consensus understanding that high levels of viral RNA production powered by 2X35S permit a more efficient initiation of virus replication. Here, we showed that the exact opposite is true. We found that, compared to the Core35S promoter, the 2X35S promoter-driven initiation of turnip crinkle virus (TCV) infection was delayed by at least 24 h. We first compared three versions of 35S promoter, namely 2X35S, 1X35S, and Core35S, for their ability to power the expression of a non-replicating green fluorescent protein (GFP) gene, and confirmed that 2X35S and Core35S correlated with the highest and lowest GFP expression, respectively. However, when inserted upstream of TCV cDNA, 2X35S-driven replication was not detected until 72 h post-inoculation (72 hpi) in inoculated leaves. By contrast, Core35S-driven replication was detected earlier at 48 hpi. A similar delay was also observed in systemically infected leaves (six versus four days post-inoculation). Combining our results, we hypothesized that the stronger 2X35S promoter might enable a higher accumulation of a TCV protein that became a repressor of TCV replication at higher cellular concentration. Extending from these results, we propose that the Core35S (or mini35S) promoter is likely a better choice for generating infectious cDNA clones of TCV.
Highlights
IntroductionPromoters are DNA sequences that are receiving increased attention as one of the primary determinants of the initiation of transcription and modulation of levels and timing of gene expression [1,2]
We investigated the strength of different versions of 35S promoter sequence for driving turnip crinkle virus (TCV) viral replicon
To elucidate the underlying mechanism of this phenomenon, here we explored the use of the core35S promoter to achieve more efficient initiation of TCV virus replication than the two other versions of 35S
Summary
Promoters are DNA sequences that are receiving increased attention as one of the primary determinants of the initiation of transcription and modulation of levels and timing of gene expression [1,2]. It controls the accurate initiation of transcription by binding to the RNA polymerase II enzyme and transcribes a gene into RNA [3]. It is usually assumed to be the key cis-acting regulatory region that controls the transcription of the adjacent coding region(s) into messenger ribonucleic acid (mRNA), which is directly translated into proteins [4]. Promoters can be identified as binding sites for trans-acting factors, “transcription factors”, which may cause activation or repression of transcription [4]
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