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

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 Erucasativa, Nasturtiumofficinale, Nicotianatabacum and Spinaciaoleracea plants and in isolated Arabidopsisthaliana 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.

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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.

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. 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).

    CAS  Article  Google Scholar 

  2. 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. 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. 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).

    CAS  Article  Google Scholar 

  5. 5.

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

    CAS  Article  Google Scholar 

  6. 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).

    CAS  Article  Google Scholar 

  7. 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).

    CAS  Article  Google Scholar 

  8. 8.

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

    CAS  Article  Google Scholar 

  9. 9.

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

    CAS  Article  Google Scholar 

  10. 10.

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

  11. 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).

    CAS  Article  Google Scholar 

  12. 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. 13.

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

    CAS  Article  Google Scholar 

  14. 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).

    CAS  Article  Google Scholar 

  15. 15.

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

    CAS  Article  Google Scholar 

  16. 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).

    CAS  Article  Google Scholar 

  17. 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).

    CAS  Article  Google Scholar 

  18. 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).

    CAS  Article  Google Scholar 

  19. 19.

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

    CAS  Article  Google Scholar 

  20. 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).

    CAS  Article  Google Scholar 

  21. 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).

    CAS  Article  Google Scholar 

  22. 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).

    CAS  Article  Google Scholar 

  23. 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).

    CAS  Article  Google Scholar 

  24. 24.

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

    CAS  Article  Google Scholar 

  25. 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).

    CAS  Article  Google Scholar 

  26. 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. 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).

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 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).

    CAS  Article  Google Scholar 

  30. 30.

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

    Article  Google Scholar 

  31. 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. 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).

    CAS  Article  Google Scholar 

  33. 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).

    CAS  Article  Google Scholar 

  34. 34.

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

    CAS  Article  Google Scholar 

  35. 35.

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

  36. 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).

    CAS  Article  Google Scholar 

  37. 37.

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

  38. 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).

    CAS  Article  Google Scholar 

  39. 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).

    CAS  Article  Google Scholar 

  40. 40.

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

    CAS  Article  Google Scholar 

  41. 41.

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

  42. 42.

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

    CAS  Article  Google Scholar 

  43. 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).

    CAS  Article  Google Scholar 

  44. 44.

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

    CAS  Article  Google Scholar 

  45. 45.

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

    CAS  Article  Google Scholar 

  46. 46.

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

    CAS  Article  Google Scholar 

  47. 47.

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

  48. 48.

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

  49. 49.

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

    CAS  Article  Google Scholar 

  50. 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).

    CAS  Article  Google Scholar 

  51. 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).

    CAS  Article  Google Scholar 

  52. 52.

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

    CAS  Article  Google Scholar 

  53. 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).

    CAS  Article  Google Scholar 

  54. 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).

    CAS  Article  Google Scholar 

  55. 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).

    CAS  Article  Google Scholar 

  56. 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).

    CAS  Article  Google Scholar 

  57. 57.

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

    CAS  Article  Google Scholar 

  58. 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. 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).

    CAS  Article  Google Scholar 

  60. 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).

    CAS  Article  Google Scholar 

  61. 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).

    CAS  Article  Google Scholar 

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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.

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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.

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Correspondence to Michael S. Strano.

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Kwak, S., 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

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