Plant Molecular Biology

, Volume 96, Issue 4–5, pp 429–443 | Cite as

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves

  • Sun Hee Rosenthal
  • Andrew G. Diamos
  • Hugh S. Mason


Key message

We have found interesting features of a plant gene (extensin) 3′ flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins.


Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3′-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.


Polyadenylation Plant based expression Tobacco extensin terminator Read-through transcription Polypurine sequence Intron 



Arabidopsis agamous


Auxiliary downstream elements


Cauliflower mosaic virus


Cleavage element


Cleavage factors


Cleavage stimulating factor


Days post infiltration


Downstream element




Far upstream element


Flowering Locus C


Green fluorescent protein


Heat shock protein


Hydroxy proline-rich glycoprotein


Intron-mediated enhancement


Near upstream element


Nopaline synthase


Norwalk virus capsid protein


Octopine synthase




Poly(A) polymerase


Polyadenylation specificity factor


Polypurine sequence


Post transcriptional gene silencing


RNA polymerase II


RNA-dependent RNA polymerase 6




Tobacco etch virus


Untranslated region


Upstream element


Vegetative storage protein





This work was supported by Grant NIH-NIAID 1 U19 AI066332-01 to H.S.M. The authors wish to thank Charles Arntzen, Yung Chang, and Tsafrir Mor for helpful discussion and advice on the manuscript.

Author contributions

SR and AD conceived the studies, conducted the experiments, analyzed the data and wrote the manuscript. HSM supervised the design of the study and data analysis and critically finalized the manuscript. The authors read and agreed to the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

11103_2018_708_MOESM1_ESM.tif (102 kb)
Fig. S1 The extensin terminator has strong activity in other plants. GFP constructs containing the extensin terminator with or without intron were agroinfiltrated into the leaves of (a) lettuce or (b) tobacco. At 4 DPI, leaf tissue was harvested and protein extracts were analyzed by SDS-PAGE followed by observation under UV illumination (365 nm) and by Coomassie staining. GFP band intensity was quantified using ImageJ software, using native plant proteins as a loading control. Columns represent means ± standard error of three independently infiltrated leaves. All leaves were infiltrated with construct pEU as an internal control for leaf and plant variability. (TIF 102 KB)
11103_2018_708_MOESM2_ESM.tif (2.6 mb)
Fig. S2 Alignment of related extensin terminators. Nucleotide sequences of homologous extensin terminators were obtained from GenBank or the Sol Genomics Network and aligned with the tobacco extensin terminator using the Clustal Omega program. Conserved features identified in this study are highlighted. (TIF 2654 KB)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Biodesign Institute, Center for Immunotherapy, Vaccines, and Virotherapy, School of Life SciencesArizona State UniversityTempeUSA

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