Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers

Nature Nanotechnology volume 14, pages 447–455 (2019)Cite this article

Subjects

Abstract

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Eruca sativa, Nasturtium officinale, Nicotiana tabacum and Spinacia oleracea plants and in isolated Arabidopsis thaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic illustration of chloroplast-targeted delivery of pDNA by using SWNTs in a plant leaf.
Fig. 2: The optimal pDNA loading on SWNTs for high-efficiency trafficking of the nanocarriers into the chloroplast.
Fig. 3: Release of DNA from DNA–chitosan-complexed SWNT conjugates in vitro and transgene expression in isolated protoplasts.
Fig. 4: Chloroplast-targeted gene delivery and transient YFP expression in mature arugula plants.
Fig. 5: Chloroplast-targeted gene delivery and transient YFP expression in mature plants.

Similar content being viewed by others

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

References

  1. Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).

    Article  CAS  Google Scholar 

  2. Abdallah, N. A., Prakash, C. S. & McHughen, A. G. Genome editing for crop improvement: challenges and opportunities. GM Crops Food 6, 183–205 (2015).

    Article  Google Scholar 

  3. Marsian, J. et al. Plant-made polio type 3 stabilized VLPs—a candidate synthetic polio vaccine. Nat. Commun. 8, 245 (2017).

    Article  Google Scholar 

  4. Li, J. F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 (2013).

    Article  CAS  Google Scholar 

  5. Yin, K., Gao, C. & Qiu, J.-L. Progress and prospects in plant genome editing. Nat. Plants 3, 17107 (2017).

    Article  CAS  Google Scholar 

  6. Duke, S. O. Perspectives on transgenic, herbicide-resistant crops in the United States almost 20 years after introduction. Pest. Manag. Sci. 71, 652–657 (2015).

    Article  CAS  Google Scholar 

  7. Fischer, R., Stoger, E., Schillberg, S., Christou, P. & Twyman, R. M. Plant-based production of biopharmaceuticals. Curr. Opin. Plant Biol. 7, 152–158 (2004).

    Article  CAS  Google Scholar 

  8. Fuentes, P., Armarego-Marriott, T. & Bock, R. Plastid transformation and its application in metabolic engineering. Curr. Opin. Biotechnol. 49, 10–15 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Maliga, P. in Genomics of Chloroplasts and Mitochondria (Advances in Photosynthesis and Respiration Series, Vol. 35) 393–414 (Springer, Dordrecht, 2012).

  11. Scott, S. E. & Wilkinson, M. J. Low probability of chloroplast movement from oilseed rape (Brassica napus) into wild Brassica rapa. Nat. Biotechnol. 17, 390–392 (1999).

    Article  CAS  Google Scholar 

  12. De Cosa, B., Moar, W., Lee, S. B., Miller, M. & Daniell, H. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 19, 71–74 (2001).

    Article  Google Scholar 

  13. Staub, J. M. et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18, 333–338 (2000).

    Article  CAS  Google Scholar 

  14. 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 

  15. Golds, T., Maliga, P. & Koop, H. U. Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Nat. Biotechnol. 11, 95–97 (1993).

    Article  CAS  Google Scholar 

  16. Cunningham, F. J., Goh, N. S., Demirer, G. S., Matos, J. L. & Landry, M. P. Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. 36, 882–897 (2018).

    Article  CAS  Google Scholar 

  17. Rafsanjani, M. S. O., Alvari, A., Samim, M., Hejazi, M. A. & Abdin, M. Z. Application of novel nanotechnology strategies in plant biotransformation: a contemporary overview. Recent Pat. Biotechnol. 6, 69–79 (2012).

    Article  CAS  Google Scholar 

  18. Ahmad, N., Michoux, F., Lössl, A. G. & Nixon, P. J. Challenges and perspectives in commercializing plastid transformation technology. J. Exp. Bot. 67, 5945–5960 (2016).

    Article  CAS  Google Scholar 

  19. Whitehead, K. A. et al. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 5, 4277 (2014).

    Article  CAS  Google Scholar 

  20. Wang, H. et al. Biocompatible chitosan–carbon dot hybrid nanogels for NIR-imaging-guided synergistic photothermal–chemo therapy. ACS Appl. Mater. Interfaces 9, 18639–18649 (2017).

    Article  CAS  Google Scholar 

  21. Deng, X. et al. Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer. Biomaterials. 35, 4333–4344 (2014).

    Article  CAS  Google Scholar 

  22. Tripathi, D. K. et al. An overview on manufactured nanoparticles in plants: uptake, translocation, accumulation and phytotoxicity. Plant Physiol. Biochem. 110, 2–12 (2017).

    Article  CAS  Google Scholar 

  23. Torney, F., Trewyn, B. G., Lin, V. S. Y. & Wang, K. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nat. Nanotechnol. 2, 295–300 (2007).

    Article  CAS  Google Scholar 

  24. Giraldo, J. P. et al. Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nat. Mater. 13, 400–408 (2014).

    Article  CAS  Google Scholar 

  25. Wong, M. H. et al. Lipid exchange envelope penetration (LEEP) of nanoparticles for plant engineering: a universal localization mechanism. Nano Lett. 16, 1161–1172 (2016).

    Article  CAS  Google Scholar 

  26. Lew, T. T. S. et al. Rational design principles for the transport and subcellular distribution of nanomaterials into plant protoplasts. Small 14, 1802086 (2018).

    Article  Google Scholar 

  27. Li, Z., de Barros, A. L. B., Soares, D. C. F., Moss, S. N. & Alisaraie, L. Functionalized single-walled carbon nanotubes: cellular uptake, biodistribution and applications in drug delivery. Int. J. Pharm. 524, 41–54 (2017).

    Article  CAS  Google Scholar 

  28. Liu, Q. et al. Carbon nanotubes as molecular transporters for walled plant cells. Nano Lett. 9, 1007–1010 (2009).

    Article  CAS  Google Scholar 

  29. Wu, Y., Phillips, J. A., Liu, H., Yang, R. & Tan, W. Carbon nanotubes protect DNA strands during cellular delivery. ACS Nano 2, 2023–2028 (2008).

    Article  CAS  Google Scholar 

  30. Malerba, M. & Cerana, R. Recent advances of chitosan applications in plants. Polymers 10, 118 (2018).

    Article  Google Scholar 

  31. Choudhary, R. C. et al. Cu-chitosan nanoparticle boost defense responses and plant growth in maize (Zea mays L.). Sci. Rep. 7, 9754 (2017).

    Article  Google Scholar 

  32. Shearer, C. J. et al. Adsorption and desorption of single-stranded DNA from single-walled carbon nanotubes. Chem. Asian J. 12, 1625–1634 (2017).

    Article  CAS  Google Scholar 

  33. Yang, Y. et al. Binding efficacy and kinetics of chitosan with DNA duplex: the effects of deacetylation degree and nucleotide sequences. Carbohydr. Polym. 169, 451–457 (2017).

    Article  CAS  Google Scholar 

  34. Jokerst, J. V., Lobovkina, T., Zare, R. N. & Gambhir, S. S. Nanoparticle PEGylation for imaging and therapy. Nanomedicine 6, 715–728 (2011).

    Article  CAS  Google Scholar 

  35. Mathur, J. & Koncz, C. in Arabidopsis Protocols (Methods in Molecular Biology Series, Vol. 82) 267–276 (Humana, Totowa, 1998).

  36. Fettiplace, R., Andrews, D. M. & Haydon, D. A. Thickness, composition and structure of some lipid bilayers and natural membranes. J. Membr. Biol. 5, 277–296 (1971).

    Article  CAS  Google Scholar 

  37. Zimmermann, U. & Neil, G. A. Electromanipulation of Cells (CRC, Boca Raton, 1996).

  38. Heikkila, E. et al. Cationic Au nanoparticle binding with plasma membrane-like lipid bilayers: potential mechanism for spontaneous permeation to cells revealed by atomistic simulations. J. Phys. Chem. C 118, 11131–11141 (2014).

    Article  CAS  Google Scholar 

  39. Wang, B., Zhang, L., Bae, S. C. & Granick, S. Nanoparticle-induced surface reconstruction of phospholipid membranes. Proc. Natl Acad. Sci. USA 105, 18171–18175 (2008).

    Article  CAS  Google Scholar 

  40. Kupiainen, M. et al. Free volume properties of sphingomyelin, DMPC, DPPC, and PLPC bilayers. J. Comput. Theor. Nanosci. 2, 401–413 (2005).

    Article  CAS  Google Scholar 

  41. Alberts, B. et al. Molecular Biology of the Cell 4th edn (Garland Science, New York, 2002).

  42. Mao, S., Sun, W. & Kissel, T. Chitosan-based formulations for delivery of DNA and siRNA. Adv. Drug Deliv. Rev. 62, 12–27 (2010).

    Article  CAS  Google Scholar 

  43. Kruss, S. et al. Neurotransmitter detection using corona phase molecular recognition on fluorescent single-walled carbon nanotube sensors. J. Am. Chem. Soc. 136, 713–724 (2014).

    Article  CAS  Google Scholar 

  44. Yang, R. et al. Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization. J. Am. Chem. Soc. 130, 8351–8358 (2008).

    Article  CAS  Google Scholar 

  45. Lesney, M. S. Polycation-like behaviour of chitosan on suspension-culture derived protoplasts of slash pine. Phytochemistry 29, 1123–1125 (1990).

    Article  CAS  Google Scholar 

  46. Kwak, S. Y. et al. A nanobionic light-emitting plant. Nano Lett. 17, 7951–7961 (2017).

    Article  CAS  Google Scholar 

  47. Díaz, A. H. & Koop, H.-U. in Chloroplast Biotechnology (Methods in Molecular Biology Series, Vol. 1132) 165–175 (Humana, Totowa, 2014).

  48. Ruhlman, T. A. in Chloroplast Biotechnology (Methods in Molecular Biology Series, Vol. 1132) 331–343 (Humana, Totowa, 2014).

  49. Asensio, J. L., Ardá, A., Cañada, F. J. & Jiménez-Barbero, J. Carbohydrate–aromatic interactions. Acc. Chem. Res. 46, 946–954 (2013).

    Article  CAS  Google Scholar 

  50. Suzuki, J. Y., Sriraman, P., Svab, Z. & Maliga, P. Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants. Plant Cell 15, 195–205 (2003).

    Article  CAS  Google Scholar 

  51. Bohmert-Tatarev, K., McAvoy, S., Daughtry, S., Peoples, O. P. & Snell, K. D. High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate. Plant Physiol. 155, 1690–1708 (2011).

    Article  CAS  Google Scholar 

  52. Thompson, C. J. et al. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J. 6, 2519–2523 (1987).

    Article  CAS  Google Scholar 

  53. 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 

  54. Herz, S., Füßl, M., Steiger, S. & Koop, H. U. Development of novel types of plastid transformation vectors and evaluation of factors controlling expression. Transgenic Res. 14, 969–982 (2005).

    Article  CAS  Google Scholar 

  55. Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907 (1987).

    Article  CAS  Google Scholar 

  56. Staub, J. M. & Maliga, P. Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J. 12, 601–606 (1993).

    Article  CAS  Google Scholar 

  57. Benfey, P. N. & Chua, N. H. Regulated genes in transgenic plants. Science 244, 174–181 (1989).

    Article  CAS  Google Scholar 

  58. van der Krol, A. R. & Chua, N. H. The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into plant nuclei. Plant Cell 3, 667–675 (1991).

    Article  Google Scholar 

  59. Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  Google Scholar 

  60. Littlejohn, G. R., Gouveia, J. D., Edner, C., Smirnoff, N. & Love, J. Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. New Phytol. 186, 1018–1025 (2010).

    Article  CAS  Google Scholar 

  61. Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G. & Citovsky, V. Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment. Nat. Protoc. 4, 71–77 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the National Research Foundation (NRF), Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) program. The Disruptive & Sustainable Technology for Agricultural Precision (DiSTAP) is an interdisciplinary research group (IRG) of the Singapore MIT Alliance for Research and Technology (SMART) Centre. We also acknowledge support of Sime Darby Malaysia. T.T.S.L. and M.H.W. were supported on a graduate fellowship by the Agency of Science, Research and Technology, Singapore. V.B.K. was supported by The Swiss National Science Foundation (project no. P300P2_174469). The authors are grateful for helpful discussion with J. P. Giraldo and thank M. M. R. Ambavaram of Yield10 Bioscience for technical guidance and C. Xu of Yield10 Bioscience (now at Jounce Therapeutics) for technical assistance.

Author information

Author notes

  1. These authors contributed equally: Seon-Yeong Kwak, Tedrick Thomas Salim Lew.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Seon-Yeong Kwak, Tedrick Thomas Salim Lew, Connor J. Sweeney, Volodymyr B. Koman, Min Hao Wong & Michael S. Strano

  2. Yield10 Bioscience, Inc., Woburn, MA, USA

    Karen Bohmert-Tatarev & Kristi D. Snell

  3. Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore

    Jun Sung Seo & Nam-Hai Chua

Authors
  1. Seon-Yeong Kwak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tedrick Thomas Salim Lew
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Connor J. Sweeney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Volodymyr B. Koman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Min Hao Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Karen Bohmert-Tatarev
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kristi D. Snell
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jun Sung Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nam-Hai Chua
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michael S. Strano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.-Y.K. and T.T.S.L. co-wrote the paper. S.-Y.K., T.T.S.L. and M.S.S. conceived and designed the experiments. C.J.S. assisted with the preparation and characterization of the chitosan-complexed SWNTs. V.B.K. performed AFM analysis. M.H.W. assisted with experimental design. K.D.S., K.B.-T. and J.S.S. constructed plasmid DNA. K.D.S. and K.B.-T. contributed the plastid DNA construct pMBX1120. J.S.S. and N.-H.C. constructed the nuclear DNA construct pBA-GFP-NL. All authors have revised the manuscript and given their approval of the final version.

Corresponding author

Correspondence to Michael S. Strano.

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 1–11

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kwak, SY., Lew, T.T.S., Sweeney, C.J. et al. Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat. Nanotechnol. 14, 447–455 (2019). https://doi.org/10.1038/s41565-019-0375-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41565-019-0375-4

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research