Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Engineered RNA-binding protein for transgene activation in non-green plastids


Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits, or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers1,2,3. Here, we report a regulatory system comprising a variant of the maize RNA-binding protein PPR10 and a cognate binding site upstream of a plastid transgene that encodes green fluorescent protein (GFP). The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulates up to 1.3% of the total soluble protein, a 60-fold increase compared with previous studies2 (0.02%). This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: PPR10 binding site system for regulating gene expression in potato amyloplasts.
Fig. 2: Leaf and tuber phenotypes of potato plants illuminated with tungsten or ultraviolet light.
Fig. 3: GFP expression in transplastomic potato plants expressing the PPR10GG protein.

Data availability

The nucleotide sequences are deposited in GenBank with accession numbers MK482729 and MK482730. Correspondence and requests for chloroplast transformation vectors and transplastomic plants should be addressed to P.M. ( Requests for plasmids encoding the PPR10 variants, and for transgenic plants expressing the PPR10 variants should be addressed to A.B. ( Biological materials will be made available pending the execution of a Materials Transfer Agreement with Rutgers University and/or the University of Oregon, as applicable.


  1. Zhang, J. et al. Identification of cis-elements conferring high levels of gene expression in non-green plastids. Plant J. 72, 115–128 (2012).

    CAS  Article  Google Scholar 

  2. Valkov, V. T. et al. High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences. Transgen. Res. 20, 137–151 (2010).

    Article  Google Scholar 

  3. Sidorov, V. A. et al. Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19, 209–216 (1999).

    CAS  Article  Google Scholar 

  4. Martin, W. et al. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl Acad. Sci. USA 99, 12246–12251 (2002).

    CAS  Article  Google Scholar 

  5. Kahlau, S. & Bock, R. Plastid transcriptomics and translatomics of tomato fruit development and chloroplast-to-chromoplast differentiation: chromoplast gene expression largely serves the production of a single protein. Plant Cell 20, 856–874 (2008).

    CAS  Article  Google Scholar 

  6. Valkov, V. T. et al. Genome-wide analysis of plastid gene expression in potato leaf chloroplasts and tuber amyloplasts: transcriptional and posttranscriptional control. Plant Physiol. 150, 2030–2044 (2009).

    CAS  Article  Google Scholar 

  7. Jin, S. & Daniell, H. The engineered chloroplast genome just got smarter. Trends Plant Sci. 20, 622–640 (2015).

    CAS  Article  Google Scholar 

  8. Zhang, J. et al. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science 347, 991–994 (2015).

    CAS  Article  Google Scholar 

  9. Boehm, C. R. & Bock, R. Recent advances and current challenges in synthetic biology of the plastid genetic system and metabolism. Plant Physiol. 179, 794–802 (2019).

    Article  Google Scholar 

  10. Maliga, P. & Bock, R. Plastid biotechnology: food, fuel and medicine for the 21st century. Plant Physiol. 155, 1501–1510 (2011).

    CAS  Article  Google Scholar 

  11. Hanson, M. R., Lin, M. T., Carmo-Silva, A. E. & Parry, M. A. Towards engineering carboxysomes into C3 plants. Plant J. 87, 38–50 (2016).

    CAS  Article  Google Scholar 

  12. Rae, B. D. et al. Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants. J. Exp. Bot. 68, 3717–3737 (2017).

    CAS  Article  Google Scholar 

  13. Long, B. M. et al. Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat. Commun. 9, 3570 (2018).

    Article  Google Scholar 

  14. Jarvis, P. & Lopez-Juez, E. Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14, 787–802 (2013).

    CAS  Article  Google Scholar 

  15. Pfalz, J., Bayraktar, O. A., Prikryl, J. & Barkan, A. Site-specific binding of a PPR protein defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts. EMBO J. 28, 2042–2052 (2009).

    CAS  Article  Google Scholar 

  16. Prikryl, J., Rojas, M., Schuster, G. & Barkan, A. Mechanism of RNA stabilization and translational activation by a pentatricopeptide repeat protein. Proc. Natl Acad. Sci. USA 108, 415–420 (2011).

    CAS  Article  Google Scholar 

  17. Barkan, A. et al. A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet. 8, e1002910 (2012).

    CAS  Article  Google Scholar 

  18. Diretto, G. et al. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS ONE 2, e350 (2007).

    Article  Google Scholar 

  19. Yu, Q., Lutz, K. A. & Maliga, P. Efficient plastid transformation in Arabidopsis. Plant Physiol. 175, 186–193 (2017).

    CAS  Article  Google Scholar 

  20. Rojas, M., Yu, Q., Williams-Carrier, R., Maliga, P. & Barkan, A. Engineered PPR proteins as inducible switches to activate the expression of chloroplast transgenes. Nat. Plants (2019).

  21. Caroca, R., Howell, K. A., Hasse, C., Ruf, S. & Bock, R. Design of chimeric expression elements that confer high-level gene activity in chromoplasts. Plant J. 73, 368–379 (2013).

    CAS  Article  Google Scholar 

  22. Slattery, C. J., Kavakli, I. H. & Okita, T. W. Engineering starch for increased quantity and quality. Trends Plant Sci. 5, 291–298 (2000).

    CAS  Article  Google Scholar 

  23. Liu, Q. et al. Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy. Plant Biotechnol. J. 15, 56–67 (2016).

    Article  Google Scholar 

  24. Miranda, R. G., Rojas, M., Montgomery, M. P., Gribbin, K. P. & Barkan, A. RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10. RNA 23, 586–599 (2017).

    CAS  Article  Google Scholar 

  25. Kuroda, H. & Maliga, P. Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol. 125, 430–436 (2001).

    CAS  Article  Google Scholar 

  26. Shinozaki, K. & Sugiura, M. Sequence of the intercistronic region between the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit and coupling factor β subunit gene. Nucleic Acids Res. 10, 4923–4934 (1982).

    CAS  Article  Google Scholar 

  27. Bevan, M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711–8721 (1984).

    CAS  Article  Google Scholar 

  28. Koncz, C. et al. in Plant Molecular Biology Manual (eds Gelvin S. B. & Schilperoort R. A.) Ch. B2, 1–22 (Kluver Academic, 1994).

  29. Svab, Z. & Maliga, P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl Acad. Sci. USA 90, 913–917 (1993).

    CAS  Article  Google Scholar 

  30. Carrer, H., Staub, J. M. & Maliga, P. Gentamycin resistance in Nicotiana conferred by AAC(3)-I, a narrow substrate specificity acetyl transferase. Plant Mol. Biol. 17, 301–303 (1991).

    CAS  Article  Google Scholar 

  31. Zommick, D. H., Kumar, G. N., Knowles, L. O. & Knowles, N. R. Translucent tissue defect in potato (Solanum tuberosum L.) tubers is associated with oxidative stress accompanying an accelerated aging phenotype. Planta 238, 1125–1145 (2013).

    CAS  Article  Google Scholar 

Download references


We thank A. Ioannou and T. Tungsuchat Huang (Rutgers University) for plastid transformation vectors pAI3 and pAI5, R. Williams-Carrier (University of Oregon) for the PPR10 antibody and Agrobacterium binary vector carrying PPR10GG under the control of a patatin promoter and F. Ludewig (University of Erlangen-Nuremberg) for the patatin promoter, T. Osumi (Simplot Plant Sciences) for sterile shoot cultures of potato cv. Desire 2-24 and R. Williams-Carrier and M. Rojas (University of Oregon) for reading the manuscript and suggestions. This research was supported by USDA NIFA Foundational Program Award Number 2014-67013-21600 to A.B. and P.M.

Author information

Authors and Affiliations



A.B. and P.M. designed the experiments. P.M. designed the plastid constructs. A.B. designed the Agrobacterium binary vector. Q.Y. transformed the potato plastid and nuclear genomes, regenerated the plants and characterized chloroplast and nuclear gene expression. Q.Y. and P.M. interpreted the results and all authors contributed to the preparation of the manuscript.

Corresponding author

Correspondence to Pal Maliga.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–3.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yu, Q., Barkan, A. & Maliga, P. Engineered RNA-binding protein for transgene activation in non-green plastids. Nat. Plants 5, 486–490 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing