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Lysozyme-coated nanoparticles for active uptake and delivery of synthetic RNA and plasmid-encoded genes in plants

Nature Plants volume 11, pages 131–144 (2025)Cite this article

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Abstract

Nanoparticle-mediated delivery of nucleic acids and proteins into intact plants has the potential to modify metabolic pathways and confer desirable traits in crops. Here we show that layered double hydroxide (LDH) nanosheets coated with lysozyme are actively taken up into the root tip, root hairs and lateral root junctions by endocytosis, and translocate via an active membrane trafficking pathway in plants. Lysozyme coating enhanced nanosheet uptake by (1) loosening the plant cell wall and (2) stimulating the expression of endocytosis and other membrane trafficking genes. The lysozyme-coated nanosheets efficiently delivered synthetic mRNA, double-stranded RNA, small interfering RNA and plasmid DNA up to 15 kb in size into tobacco roots, and also functional nucleic acids into leaves, callus, flowers and developing pollen of dicot and monocot species. Thus, lysozyme-coated LDH nanoparticles are a versatile tool for efficiently delivering functional nucleic acids into plants.

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Fig. 1: Characterization of LDH nanoparticles.
Fig. 2: Uptake of LDH nanoparticles in N. benthamiana roots.
Fig. 3: Active uptake and translocation of lysozyme-coated LDH30 nanoparticles.
Fig. 4: Lysozyme-coated LDH30-mediated delivery of synthetic GFP mRNA into roots of four plant species and sorghum callus.
Fig. 5: Lysozyme-coated LDH30 nanoparticle delivery of synthetic dsRNA, siRNA and plasmid DNA into roots, leaves and pollen.
Fig. 6: Schematic model of lysozyme-coated LDH nanocarrier uptake, translocation, cellular internalization and delivery of nucleic acids in the root.

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Data availability

There are no restrictions on data availability. All data supporting this research are included in the main text, figures and extended data figures, and supplementary documents. Nucleic acid sequences and plasmid maps are provided in supporting datasets of the paper. Source data for figures and original gel images are provided with this paper.

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Acknowledgements

We acknowledge the support from the Australian Research Council Discovery Project (DP190103486) and the Research Hub (IH190100022), and from the National Health and Medical Research Council (APP1175808); the facilities, and the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis and the Australian National Fabrication Facility (ANFF, Qld Node), The University of Queensland, and the microscopy facility at the Institute for Molecular Bioscience, The University of Queensland.

Author information

Author notes

  1. Neena Mitter

    Present address: Charles Sturt University, Wagga Wagga, New South Wales, Australia

Authors and Affiliations

  1. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia

    Jiaxi Yong, Miaomiao Wu, Run Zhang & Zhi Ping Xu

  2. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

    Jiaxi Yong, Wang Xu, Christopher W. G. Mann & Bernard J. Carroll

  3. Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia

    Run Zhang

  4. Centre for Crop Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia

    Guoquan Liu

  5. Centre for Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Australia

    Christopher A. Brosnan & Neena Mitter

  6. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China

    Zhi Ping Xu

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Contributions

J.Y., R.Z., B.J.C. and Z.P.X. conceptualized and designed the research. J.Y. synthesized nanoparticles, conducted uptake and delivery experiments and data analysis. W.X. contributed to plasmid delivery experiments, endocytosis experiments and qPCR. M.W. synthesized and characterized nanoparticles and contributed to the schematic illustrations. C.W.G.M. produced the dsRNA, G.L. prepared the pUBI:GFP plasmid and sorghum callus, and C.A.B. contributed to western blots. C.A.B. and N.M. contributed reagents and advice. J.Y. wrote the first draft of the paper, and J.Y., B.J.C. and Z.P.X. edited and revised the paper. All authors read and commented on the paper.

Corresponding authors

Correspondence to Bernard J. Carroll or Zhi Ping Xu.

Ethics declarations

Competing interests

N.M., B.J.C. and Z.P.X. have received research grant funding from industry partners, which involves commercialization activities related to the LDH nanoparticles. Z.P.X., B.J.C., N.M., J.Y. and W.X. are inventors on an international patent filed by UniQuest that describes protein-coated LDH nanoparticles for nucleic acid delivery into plants (PCT/AU2024/050554). The other authors declare no competing interests.

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Nature Plants thanks Karl-Heinz Kogel, Markita Landry and Yan Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Protein coating of the LDH30 nanosheets and the addition of 20 mM KCl to the hydroponics both suppress the aggregation of nanoparticles on the root surface.

A, Congo red-labelled, uncoated LDH nanoparticle aggregates on A. thaliana root surfaces after 12 h incubation (200 mg/L of LDH nanoparticles) but BSA-coated Congo red-labelled LDH nanoparticles did not. Bar = 200 µm. Three biological replicates were analysed with similar results. B, Alleviation of aggregation of fluorescein-labelled LDH nanoparticles (LDH30-FL) in the root incubation media by the addition of 20 mM KCl and/or lysozyme and bovine serum albumin (BSA) coating of the nanoparticles but not by the addition of 20 mM MgCl2. Orange/red clusters in the suspension are caused by aggregated particles.

Extended Data Fig. 2 Z-stacked confocal images showing internalization and translocation of lysozyme-coated LDH30 labelled with fluorescein in a N. benthamiana root at 4 h post treatment.

Bar = 100 µm.

Extended Data Fig. 3 Lysozyme enzyme activity is required for enhancing LDH30 uptake.

A, Representative confocal images of N. benthamiana roots showing that a 4 h pre-treatment with 1 mg/ml of imidazome-inhibited lysozyme failed to enhance uptake of uncoated LDH30 nanoparticles loaded with fluorescein (LDH30-FL), and comparison to roots pre-treated with lysozyme or treated with lysozyme-coated LDH nanoparticles (Lys@LDH30-FL). Bar = 200 µm. B, Quantitative fluorescence analysis showing pre-treatment with imidazole-inhibited lysozyme did not enhance uptake of LDH30-FL (mean ± SEM for n = 9 biological replicates). Treatments labelled with the same letter in B were not significantly different (p < 0.05) based on one-way ANOVA with post-hoc Tukey’s HSD test.

Source data

Extended Data Fig. 4 Lysozyme enhances LDH30 uptake by tomato early bicellular pollen.

A, Representative confocal images of early bicellular tomato pollen showing that a 4 h pre-treatment with 1 mg/ml lysozyme enhances uptake of uncoated LDH30 nanoparticles loaded with fluorescein. B, Flow cytometry data showing enhanced uptake of LDH30 labelled with fluorescein (LDH30-FL) by early bicellular pollen after lysozyme pre-treatment (Lysozyme then LDH30-FL) or lysozyme coating of the nanosheets (Lys@LDH30-FL). C, Flow cytometry data showing pre-treatment or nanosheet coating with heat treated lysozyme (HT-Lysozyme/HT-Lys) failed to enhance uptake of LDH30-FL by pollen. The fluorescence intensity was normalized based on the auto-fluorescence of pollen in the untreated control group. Treatments labelled with the same letter in B and C were not significantly different (p < 0.05) based on one-way ANOVA with post-hoc Tukey’s HSD test. Data in B and C are presented as the mean ± SEM for n = 3 biological replicates.

Source data

Extended Data Fig. 5 Cellulase pretreatment enhances the uptake of LDH30 in mature roots but not in the root tip.

Representative confocal images (A) and quantification of fluorescein intensity (B) of roots pre-treated for 4 h with cellulase or lysozyme prior to treatment with uncoated LDH30 nanoparticles loaded with fluorescein (LDH30-FL; mean ± SEM for n = 3 biological replicates). Bar = 100 µm. Treatments labelled with the same letter in B were not significantly different (p < 0.05) based on one-way ANOVA with post-hoc Tukey’s HSD test.

Source data

Extended Data Fig. 6 Uptake and translocation of lysozyme-coated LDH30 nanosheets labelled with fluorescein from roots into hypocotyls.

A, Time-course images showing nanosheet uptake in three N. benthamiana seedlings; dash white line indicates the junction of the root and hypocotyl. Bar = 1 cm. B, Zoomed in images of hypocotyls in A. Bar = 0.25 cm. C, Green channel fluorescence intensity analysis of hypocotyls in panel B at 0.5 cm above the root-hypocotyl junction (mean ± SEM for n = 3 biological replicates).

Extended Data Fig. 7 Expression levels of endocytosis and membrane trafficking genes post incubation with lysozyme, high temperature-treated lysozyme and BSA.

RT-qPCR analysis of mRNA levels of endocytosis and membrane trafficking genes in Arabidopsis roots at 8 h post incubation with lysozyme, high temperature-treated lysozyme and BSA. Data are presented as the mean ± SEM for n = 3 biological replicates. Treatments labelled with the same letter in the figure were not significantly different (p < 0.05) based on two-way ANOVA with post-hoc Tukey’s HSD test.

Source data

Extended Data Fig. 8 Lysozyme-coated LDH nanoparticles deliver GFP mRNA into sorghum callus.

Camera images (A, bar = 1 cm) and quantitative analysis of green channel pixel intensities of sorghum calli (B) at day 2 post treatment with lysozyme-coated LDH30 loaded with GFP mRNA (Lys@LDH30-mRNA) (mean ± SEM for n = 5 biological replicates; *, p = 0.0136 based on a two-tailed t-test). Bar = 1 cm. The concentration of mRNA in the treatment solution was 5 mg/L.

Source data

Extended Data Fig. 9 Delivery and expression of a 35S:GFP gene in N. benthamiana roots.

Representative confocal images showing GFP expression pattern in mature root and root tip at day 2 post treatment with lysozyme-coated LDH30 loaded with a 6.1 kb plasmid encoding 35S:GFP (Lys@LDH30-pDNA-1). Bar = 20 µm.

Extended Data Fig. 10 Lysozyme-coated LDH30 delivery and expression of 6.1 kb and 14.7 kb plasmids encoding the same 35S:GFP gene.

A, Representative confocal microscope images of N. benthamiana roots at day 2 post incubation with lysozyme-coated LDH30 loaded with a 14.7 kb plasmid encoding a 35S:GFP gene (Lys@LDH30-pDNA-2) at a plasmid DNA concentration of 10 mg/L. B, Comparison of GFP fluorescence intensity in roots incubated with Lys@LDH30-pDNA-2 or lysozyme-coated LDH30 loaded with a 6.1 kb plasmid encoding the same 35S:GFP gene (Lys@LDH30-pDNA-1) (mean ± SEM for n = 9 biological replicates). Data were normalized relative to control roots treated with lysozyme-coated LDH30 without plasmid DNA (Lys@LDH30). Treatments labelled with the same letter in B were not significantly different (p < 0.05) based on one-way ANOVA with post-hoc Tukey’s HSD test.

Source data

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Supplementary Figs. 1–18, Tables 1 and 2, Datasets 1–7 and Fig. 1.

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Yong, J., Xu, W., Wu, M. et al. Lysozyme-coated nanoparticles for active uptake and delivery of synthetic RNA and plasmid-encoded genes in plants. Nat. Plants 11, 131–144 (2025). https://doi.org/10.1038/s41477-024-01882-x

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