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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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
Zhang, J. et al. Identification of cis-elements conferring high levels of gene expression in non-green plastids. Plant J. 72, 115–128 (2012).
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).
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).
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).
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).
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).
Jin, S. & Daniell, H. The engineered chloroplast genome just got smarter. Trends Plant Sci. 20, 622–640 (2015).
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).
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).
Maliga, P. & Bock, R. Plastid biotechnology: food, fuel and medicine for the 21st century. Plant Physiol. 155, 1501–1510 (2011).
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).
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).
Long, B. M. et al. Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat. Commun. 9, 3570 (2018).
Jarvis, P. & Lopez-Juez, E. Biogenesis and homeostasis of chloroplasts and other plastids. Nat. Rev. Mol. Cell Biol. 14, 787–802 (2013).
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).
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).
Barkan, A. et al. A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet. 8, e1002910 (2012).
Diretto, G. et al. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway. PLoS ONE 2, e350 (2007).
Yu, Q., Lutz, K. A. & Maliga, P. Efficient plastid transformation in Arabidopsis. Plant Physiol. 175, 186–193 (2017).
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).
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).
Slattery, C. J., Kavakli, I. H. & Okita, T. W. Engineering starch for increased quantity and quality. Trends Plant Sci. 5, 291–298 (2000).
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).
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).
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).
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).
Bevan, M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 8711–8721 (1984).
Koncz, C. et al. in Plant Molecular Biology Manual (eds Gelvin S. B. & Schilperoort R. A.) Ch. B2, 1–22 (Kluver Academic, 1994).
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).
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).
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).
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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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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DOI: https://doi.org/10.1038/s41477-019-0413-0
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