High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants

Abstract

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis and crop engineering. The plant cell wall is a barrier that limits the ease and throughput of exogenous biomolecule delivery to plants. Current delivery methods either suffer from host-range limitations, low transformation efficiencies, tissue damage or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into intact plants of several species with pristine and chemically functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.

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Fig. 1: Strategies for grafting DNA on CNT scaffolds and characterization of DNA–CNT conjugates.
Fig. 2: DNA delivery into mature plant leaves with CNTs and subsequent GFP expression.
Fig. 3: Transient CNT-mediated GFP expression in mature plant leaves and nanoparticle toxicity assessment.
Fig. 4: DNA delivery into isolated protoplasts with CNTs and subsequent GFP expression.

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.

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Acknowledgements

The authors acknowledge support from a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Stanley Fahn PDF Junior Faculty Grant under award no. PF-JFA-1760, a Beckman Foundation Young Investigator Award, a USDA AFRI award, a grant from the Gordon and Betty Moore Foundation, a USDA NIFA award, support from the Chan-Zuckerberg foundation and an FFAR New Innovator Award (to M.P.L). G.S.D. is supported by a Schlumberger Foundation Faculty for the Future Fellowship. L.C. is supported by National Defense Science and Engineering Graduate (NDSEG) Fellowship and by the LAM Foundation. The authors thank C. Gee for assisting with the Imaging-PAM Maxi fluorimeter, A. Schultink and A. Ortega for helpful discussions and C. Jakobson and D. Tullman-Ercek for generously sharing their laboratory resources. The authors also acknowledge support from UC Berkeley Molecular Imaging Center (supported by the Gordon and Betty Moore Foundation), the UC Berkeley Biological Imaging Facility (supported in part by the National Institutes of Health S10 program under award no. 1S10OD018136-01), the QB3 Shared Stem Cell Facility, the Innovative Genomics Institute (IGI), and R. Zalpuri at the Electron Microscopy Lab at UC Berkeley for TEM sample preparation and imaging.

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G.S.D. and M.P.L. conceived of the project, designed the study and wrote the manuscript. G.S.D. performed the majority of experiments and all data analysis. H.Z. and L.C. performed AFM imaging, and H.Z. also performed nanoparticle internalization experiments into mature leaves and western blot experiments. J.L.M. performed Agrobacterium and wheat transformation experiments. N.S.G. helped with designing ddPCR experiments and performed CNT leaf toxicity confocal imaging and TIRF experiments. F.C. performed nanoparticle internalization experiments into isolated protoplasts. Y.S. performed TEM imaging of leaves. A.J.A. and R.C. prepared the plasmids used in the studies. M.-J.C. performed particle bombardment experiments. All authors have edited and commented on the manuscript, and have given their approval of the final version.

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Correspondence to Markita P. Landry.

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Journal peer review information: Nature Nanotechnology thanks Neena Mitter, Eleni Stavrinidou and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Demirer, G.S., Zhang, H., Matos, J.L. et al. High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants. Nat. Nanotechnol. 14, 456–464 (2019). https://doi.org/10.1038/s41565-019-0382-5

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