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
Article
First Online:
Received:
Accepted:

Abstract

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.

Abstract

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.

Keywords

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

Abbreviations

AG

Arabidopsis agamous

auxDSE

Auxiliary downstream elements

CaMV

Cauliflower mosaic virus

CE

Cleavage element

CF

Cleavage factors

CstF

Cleavage stimulating factor

DPI

Days post infiltration

DSE

Downstream element

Ext

Extensin

FUE

Far upstream element

FLC

Flowering Locus C

GFP

Green fluorescent protein

HSP

Heat shock protein

HRGP

Hydroxy proline-rich glycoprotein

IME

Intron-mediated enhancement

NUE

Near upstream element

NOS

Nopaline synthase

NVCP

Norwalk virus capsid protein

OCS

Octopine synthase

poly(A)

Polyadenylation

PAP

Poly(A) polymerase

CPSF

Polyadenylation specificity factor

PPS

Polypurine sequence

PTGS

Post transcriptional gene silencing

RNAPII

RNA polymerase II

RDR6

RNA-dependent RNA polymerase 6

STK

Seedstick

TEV

Tobacco etch virus

UTR

Untranslated region

USE

Upstream element

VSP

Vegetative storage protein

GUS

β-Glucuronidase

This is a preview of subscription content, log in to check access

Notes

Acknowledgements

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)

References

  1. Ali S, Taylor WC (2001) The 3′ non-coding region of a C4 photosynthesis gene increases transgene expression when combined with heterologous promoters. Plant Mol Biol 46:325–333CrossRefPubMedGoogle Scholar
  2. Arntzen CJ (2008) Plant science. Using tobacco to treat cancer. Science 321:1052–1053.  https://doi.org/10.1126/science.1163420 CrossRefPubMedGoogle Scholar
  3. Baeg K, Iwakawa H, Tomari Y (2017) The poly(A) tail blocks RDR6 from converting self mRNAs into substrates for gene silencing. Nat Plants 3:17036.  https://doi.org/10.1038/nplants.2017.36 CrossRefPubMedGoogle Scholar
  4. Beyene G, Buenrostro-Nava MT, Damaj MB, Gao S-J, Molina J, Mirkov TE (2011) Unprecedented enhancement of transient gene expression from minimal cassettes using a double terminator. Plant Cell Rep 30:13–25.  https://doi.org/10.1007/s00299-010-0936-3 CrossRefPubMedGoogle Scholar
  5. Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamianato enhance molecular plant-microbe biology research. Mol Plant-Microbe Interact 25:1523–1530.  https://doi.org/10.1094/mpmi-06-12-0148-ta CrossRefPubMedGoogle Scholar
  6. Carrington JC, Freed DD, Leinicke AJ (1991) Bipartite signal sequence mediates nuclear translocation of the plant Potyviral NIa. Protein The Plant Cell 3:953.  https://doi.org/10.2307/3869157 PubMedGoogle Scholar
  7. Chung BYW, Simons C, Firth AE, Brown CM, Hellens RP (2006) Effect of 5’UTR introns on gene expression in Arabidopsis thaliana. BMC Genom 7:120.  https://doi.org/10.1186/1471-2164-7-120 CrossRefGoogle Scholar
  8. Collens JI, Mason HS, Curtis WR (2007) Agrobacterium-mediated viral vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture. Biotechnol Prog 23:570–576.  https://doi.org/10.1021/bp060342u CrossRefPubMedGoogle Scholar
  9. Dantonel J-C, Murthy KGK, Manley JL, Tora L (1997) Transcription factor TFIID recruits factor CPSF for formation of 3′ end of mRNA. Nature 389:399–402.  https://doi.org/10.1038/38763 CrossRefPubMedGoogle Scholar
  10. Diamos AG, Rosenthal SH, Mason HS (2016) 5′ and 3′ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves. Front Plant Sci.  https://doi.org/10.3389/fpls.2016.00200 PubMedPubMedCentralGoogle Scholar
  11. Dong H et al (2007) An exploration of 3′-end processing signals and their tissue distribution in Oryza sativa. Gene 389:107–113.  https://doi.org/10.1016/j.gene.2006.10.015 CrossRefPubMedGoogle Scholar
  12. Francis KE, Spiker S (2005) Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant J 41:464–477.  https://doi.org/10.1111/j.1365-313x.2004.02312.x CrossRefPubMedGoogle Scholar
  13. Gilmartin GM (2005) Eukaryotic mRNA 3′ processing: a common means to different ends. Genes Dev 19:2517–2521.  https://doi.org/10.1101/gad.1378105 CrossRefPubMedGoogle Scholar
  14. Gleba YY, Tusé D, Giritch A (2014) Plant viral vectors for delivery by Agrobacterium. In: Current topics in microbiology and immunology. pp 155–192Google Scholar
  15. Gray NK, Hentze MW (1994) Regulation of protein synthesis by mRNA structure. Mol Biol Rep 19:195–200.  https://doi.org/10.1007/bf00986961 CrossRefPubMedGoogle Scholar
  16. Hirsinger C, Parmentier Y, Durr A, Fleck J, Jamet E (1997) Characterization of a tobacco extensin gene and regulation of its gene family in healthy plants and under various stress conditions. Plant Mol Biol 33:279–289.  https://doi.org/10.1023/a:1005738815383 CrossRefPubMedGoogle Scholar
  17. Huang Z, Mason HS (2004) Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system. Plant Biotechnol J 2:241–249.  https://doi.org/10.1111/j.1467-7652.2004.00068.x CrossRefPubMedGoogle Scholar
  18. Hunt AG (2008) Messenger RNA 3′-end formation in plants. Curr Top Microbiol Immunol 326:151–177.  https://doi.org/10.1007/978-3-540-76776-3_9 PubMedGoogle Scholar
  19. Ingelbrecht LW, Herman LMF, Dekeyser RA, Montagu MC, Van Depicker AG (1989) Different 3′-end regions strongly lnfluence the leve1 of gene expression in plant cells. Plant Cell 1:671–680.  https://doi.org/10.1105/tpc.1.7.671 PubMedPubMedCentralGoogle Scholar
  20. Ji G, Zheng J, Shen Y, Wu X, Jiang R, Lin Y, Loke JC, Davis KM, Reese GJ, Li QQ (2007) Predictive modeling of plant messenger RNA polyadenylation sites. BMC Bioinform 8:43.  https://doi.org/10.1186/1471-2105-8-43 CrossRefGoogle Scholar
  21. Jiang X, Wang M, Graham DY, Estes MK (1992) Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J Virol 66:6527–6532PubMedPubMedCentralGoogle Scholar
  22. Kato H, Xie G, Sato Y, Imai R (2010) Isolation of anther-specific gene promoters suitable for transgene expression in rice. Plant Mol Biol Rep 28:381–387.  https://doi.org/10.1007/s11105-009-0162-8 CrossRefGoogle Scholar
  23. Kertész S, Kerényi Z, Mérai Z, Bartos I, Pálfy T, Barta E, Silhavy D (2006) Both introns and long 3′-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants. Nucleic Acids Res 34:6147–6157.  https://doi.org/10.1093/nar/gkl737 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kooiker M, Airoldi CA, Losa A, Manzotti PS, Finzi L, Kater MM, Colombo L (2005) BASIC PENTACYSTEINE1, a GA binding protein that induces conformational changes in the regulatory region of the homeotic Arabidopsis gene SEEDSTICK. Plant Cell 17:722–729.  https://doi.org/10.1105/tpc.104.030130 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Libri D, Dower K, Boulay J, Thomsen R, Rosbash M, Jensen TH (2002) Interactions between mRNA export commitment, 3′-end quality control, and nuclear degradation. Mol Cell Biol 22:8254–8266.  https://doi.org/10.1128/MCB.22.23.8254-8266.2002 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Loke JC (2005) Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures. Plant Physiol 138:1457–1468.  https://doi.org/10.1104/pp.105.060541 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lomonossoff GP, D’Aoust M-A (2016) Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Science 353:1237–1240.  https://doi.org/10.1126/science.aaf6638 CrossRefPubMedGoogle Scholar
  28. Luo Z, Chen Z (2007) Improperly terminated, unpolyadenylated mRNA of sense transgenes is targeted by RDR6-mediated RNA silencing in Arabidopsis. Plant Cell 19:943–958.  https://doi.org/10.1105/tpc.106.045724 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Mandel CR, Bai Y, Tong L (2008) Protein factors in pre-mRNA 3′-end processing cellular and. Mol Life Sci 65:1099–1122.  https://doi.org/10.1007/s00018-007-7474-3 CrossRefGoogle Scholar
  30. Mapendano CK, Lykke-Andersen S, Kjems J, Bertrand E, Jensen TH (2010) Crosstalk between mRNA 3′-end processing and transcription initiation. Mol Cell 40:410–422.  https://doi.org/10.1016/j.molcel.2010.10.012 CrossRefPubMedGoogle Scholar
  31. Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci 93:5335–5340.  https://doi.org/10.1073/pnas.93.11.5335 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Mathew LG, Maloney B, Takeda N, Mason HS (2011) Spurious polyadenylation of Norovirus Narita 104 capsid protein mRNA in transgenic plants. Plant Mol Biol 75:263–275.  https://doi.org/10.1007/s11103-010-9725-1 CrossRefPubMedGoogle Scholar
  33. Menossi M, Rabaneda F, Puigdomenech P, Martinez-Izquierdo JA (2003) Analysis of regulatory elements of the promoter and the 3′ untranslated region of the maize Hrgp gene coding for a cell wall protein. Plant Cell Rep 21:916–923.  https://doi.org/10.1007/s00299-003-0602-0 PubMedGoogle Scholar
  34. Millevoi S, Vagner S (2010) Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation. Nucleic Acids Res 38:2757–2774.  https://doi.org/10.1093/nar/gkp1176 CrossRefPubMedGoogle Scholar
  35. Mogen BD, MacDonald MH, Graybosch R, Hunt AG (1991) Upstream sequences other than AAUAAA are required for efficient messenger RNA 3′-end formation in plants. Plant Cell 2:1261.  https://doi.org/10.2307/3869344 Google Scholar
  36. Moore MJ, Proudfoot NJ (2009) Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136:688–700.  https://doi.org/10.1016/j.cell.2009.02.001 CrossRefPubMedGoogle Scholar
  37. Mor TS, Moon Y-S, Palmer KE, Mason HS (2003) Geminivirus vectors for high-level expression of foreign proteins in plant cells. Biotechnol Bioeng 81:430–437.  https://doi.org/10.1002/bit.10483 CrossRefPubMedGoogle Scholar
  38. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328–332.  https://doi.org/10.1093/pcp/pcp188 CrossRefPubMedGoogle Scholar
  39. Narsai R, Howell KA, Millar AH, O’Toole N, Small I, Whelan J (2007) Genome-wide analysis of mRNA decay rates and their determinants in Arabidopsis thaliana. Plant Cell Online 19:3418–3436.  https://doi.org/10.1105/tpc.107.055046 CrossRefGoogle Scholar
  40. Qu X, Lykke-Andersen S, Nasser T, Saguez C, Bertrand E, Jensen TH, Moore C (2009) Assembly of an export-competent mRNP Is needed for efficient release of the 3′-end processing complex after polyadenylation. Mol Cel Biol 29:5327–5338.  https://doi.org/10.1128/mcb.00468-09 CrossRefGoogle Scholar
  41. Rethmeier N, Seurinck J, Van Montagu M, Cornelissen M (1997) Intron-mediated enhancement of transgene expression in maize is a nuclear, gene-dependent process. Plant J 12:895–899.  https://doi.org/10.1046/j.1365-313x.1997.12040895.x CrossRefPubMedGoogle Scholar
  42. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS (2000) Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol 18:1167–1171.  https://doi.org/10.1038/81153 CrossRefPubMedGoogle Scholar
  43. Rose AB (2008) Intron-mediated regulation of gene expression. In: Nuclear pre-mRNA processing in plants. Springer, HeidelbergGoogle Scholar
  44. Rothnie HM, Chen G, Futterer J, Hohn T (2001) Polyadenylation in rice Tungro bacilliform virus: cis-acting signals and regulation. J Virol 75:4184–4194.  https://doi.org/10.1128/jvi.75.9.4184-4194.2001 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sanfacon H, Brodmann P, Hohn T (1991) A dissection of the cauliflower mosaic virus polyadenylation signal. Genes Dev 5:141–149.  https://doi.org/10.1101/gad.5.1.141 CrossRefPubMedGoogle Scholar
  46. Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832.  https://doi.org/10.1016/j.biotechadv.2009.06.004 CrossRefPubMedGoogle Scholar
  47. Sheldon CC, Conn AB, Dennis ES, Peacock WJ (2002) Different regulatory regions are required for the vernalization-induced repression of Flowering Locus C and for the epigenetic maintenance of repression. Plant Cell Online 14:2527–2537.  https://doi.org/10.1105/tpc.004564 CrossRefGoogle Scholar
  48. Showalter AM, Keppler B, Lichtenberg J, Gu D, Welch LR (2010) A bioinformatics approach to the identification, classification, and analysis of hydroxyproline-rich Glycoproteins. Plant Physiol 153:485–513.  https://doi.org/10.1104/pp.110.156554 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Sieburth LE, Meyerowitz EM (1997) Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355.  https://doi.org/10.2307/3870487 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Slomovic S, Schuster G (2013) Circularized RT-PCR (cRT-PCR): analysis of the 5′ ends, 3′ ends, and poly (A) tails of RNA. In: Methods in enzymology, vol 530, Academic Press, Elsevier, pp 227–225CrossRefGoogle Scholar
  51. Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15.  https://doi.org/10.1111/j.1467-7652.2006.00216.x CrossRefPubMedGoogle Scholar
  52. Tan-Wong SM, Wijayatilake HD, Proudfoot NJ (2009) Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex. Genes Dev 23:2610–2624.  https://doi.org/10.1101/gad.1823209 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Tan-Wong SM et al (2012) Gene loops enhance transcriptional directionality. Science 338:671–675.  https://doi.org/10.1126/science.1224350 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Thanavala Y, Huang Z, Mason HS (2006) Plant-derived vaccines: a look back at the highlights and a view to the challenges on the road ahead. Expert Rev Vaccine 5:249–260.  https://doi.org/10.1586/14760584.5.2.249 CrossRefGoogle Scholar
  55. Tian B, Manley JL (2013) Alternative cleavage and polyadenylation: the long and short of it. Trends Biochem Sci 38:312–320.  https://doi.org/10.1016/j.tibs.2013.03.005 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Xing A, Moon BP, Mills KM, Falco SC, Li Z (2010) Revealing frequent alternative polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators. Plant Biotechnol J 8:772–782.  https://doi.org/10.1111/j.1467-7652.2010.00504.x CrossRefPubMedGoogle Scholar
  57. Zhang X, Mason H (2006) Bean yellow dwarf virus replicons for high-level transgene expression in transgenic plants and cell cultures. Biotechnol Bioeng 93:271–279.  https://doi.org/10.1002/bit.20695 CrossRefPubMedGoogle Scholar
  58. Zhao J, Hyman L, Moore C (1999) Formation of mRNA 3′-ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63:405–445PubMedPubMedCentralGoogle Scholar
  59. Zhao H, Xing D, Li QQ (2009) Unique features of plant cleavage and polyadenylation specificity factor revealed by proteomic studies. Plant Physiol 151:1546–1556.  https://doi.org/10.1104/pp.109.142729 CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations