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

Abstract

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.

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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.

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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. (maliga@waksman.rutgers.edu). Requests for plasmids encoding the PPR10 variants, and for transgenic plants expressing the PPR10 variants should be addressed to A.B. (abarkan@uoregon.edu). 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.

References

  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  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).

    Article  CAS  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 https://doi.org/10.1038/s41477-019-0412-1 (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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  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).

    Article  CAS  Google Scholar 

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Acknowledgements

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.

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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.

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Correspondence to Pal Maliga.

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Yu, Q., Barkan, A. & Maliga, P. Engineered RNA-binding protein for transgene activation in non-green plastids. Nat. Plants 5, 486–490 (2019). https://doi.org/10.1038/s41477-019-0413-0

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