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Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses

Nature Plants volume 3, Article number: 16207 (2017) Cite this article

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Abstract

Topical application of pathogen-specific double-stranded RNA (dsRNA) for virus resistance in plants represents an attractive alternative to transgenic RNA interference (RNAi). However, the instability of naked dsRNA sprayed on plants has been a major challenge towards its practical application. We demonstrate that dsRNA can be loaded on designer, non-toxic, degradable, layered double hydroxide (LDH) clay nanosheets. Once loaded on LDH, the dsRNA does not wash off, shows sustained release and can be detected on sprayed leaves even 30 days after application. We provide evidence for the degradation of LDH, dsRNA uptake in plant cells and silencing of homologous RNA on topical application. Significantly, a single spray of dsRNA loaded on LDH (BioClay) afforded virus protection for at least 20 days when challenged on sprayed and newly emerged unsprayed leaves. This innovation translates nanotechnology developed for delivery of RNAi for human therapeutics to use in crop protection as an environmentally sustainable and easy to adopt topical spray.

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Figure 1: Characterization of LDH nanosheets and dsRNA loading into LDH.
Figure 2: Breakdown of LDH and release of dsRNA, and uptake of dsRNA and induction of RNAi, in transgenic Arabidopsis seedlings.
Figure 3: Adherence and stability of dsRNA loaded into LDH.
Figure 4: BioClay (dsRNA–LDH) spray provides protection against viruses in local lesion assays.
Figure 5: Topical application of BioClay (dsRNA–LDH) affords prolonged and systemic protection from CMV in N. tabacum.
Figure 6: CMV2b-BioClay and CMV2b-dsRNA application suppresses the vsiRNA load in N. tabacum in response to CMV inoculation.

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Acknowledgements

This work was supported by the Bill and Melinda Gates Foundation Grand Challenges Exploration Grant and the University of Queensland's Collaborative Industry Engagement Fund followed by Accelerated Partnership Grant, Queensland Government awarded to N.M. and the ARC Future Fellowship (FT120100813) awarded to Z.P.X. Special thanks goes to M. Pointon and B. Duggan from Nufarm Australia Ltd as the industry partner and D. Ferguson from Uniquest, the commercialization arm of the University of Queensland for support and suggestions. We thank K. Vinall for technical assistance with the confocal microscopy studies. The PMMoVIR54 construct was a kind gift provided by F. Tenllado, Centro de Investigaciones Biológicas, Madrid, Spain. E.A.W. PhD programme with N.M. is supported by a scholarship from the University of Queensland.

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Authors and Affiliations

  1. Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Brisbane, 4072, Queensland, Australia

    Neena Mitter, Elizabeth A. Worrall, Karl E. Robinson, Ritesh G. Jain, Christelle Taochy & Stephen J. Fletcher

  2. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, 4072, Queensland, Australia

    Peng Li, G. Q. (Max) Lu & Zhi Ping Xu

  3. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, 4072, Queensland, Australia

    Christelle Taochy, Stephen J. Fletcher & Bernard J. Carroll

  4. University of Surrey, Guildford, GU2 7XH, UK

    G. Q. (Max) Lu

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  1. Neena Mitter
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Contributions

N.M., Z.P.X. and G.Q.L. conceived the BioClay technology. N.M., E.A.W., K.E.R., B.J.C. and Z.P.X. wrote the manuscript. N.M., Z.P.X. and B.J.C. provided expertise and supervised the work. E.A.W. preformed the experiments on constructs and expression of dsRNA, dsRNA loading into LDH, breakdown of LDH and release of dsRNA, stability of dsRNA bound to LDH, dsRNA uptake studies and crop protection assays. K.E.R. preformed experiments on constructs and expression of dsRNA, dsRNA loading into LDH, stability of dsRNA bound to LDH and crop protection assays. P.L. preformed experiments on LDH synthesis and characterization. R.G.J. preformed experiments on the northern blot detection of dsRNA uptake. C.T. preformed experiments on the GUS reporter system. S.J.F. preformed all statistical and bioinformatics analyses. All authors edited the manuscript.

Corresponding authors

Correspondence to Neena Mitter or Zhi Ping Xu.

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The authors declare no competing financial interests.

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Supplementary Information

Supplementary Figures 1–11, Supplementary Tables 1 and 2. (PDF 1674 kb)

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Mitter, N., Worrall, E., Robinson, K. et al. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants 3, 16207 (2017). https://doi.org/10.1038/nplants.2016.207

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