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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

    Flood, J. The importance of plant health to food security. Food Secur. 2, 215–231 (2010).

    Article  Google Scholar 

  2. 2

    Bebber, D. P., Ramotowski, M. A. T. & Gurr, S. J. Crop pests and pathogens move polewards in a warming world. Nat. Clim. Change 3, 985–988 (2013).

    Article  Google Scholar 

  3. 3

    Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Bartel, D. P. MicroRNAs: genomics, biogensis, mechanism and function. Cell 116, 281–297 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Burand, J. P. & Hunter, W. B. RNAi: future in insect management. J. Invertebr. Pathol. 112, S68–S74 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Mailard, P. V. et al. Antiviral RNA interference in mammalian cells. Science 342, 235–238 (2013).

    Article  Google Scholar 

  7. 7

    Borges, F. & Martienssen, R. A. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16, 727–741 (2015).

    CAS  Article  Google Scholar 

  8. 8

    Gordon, K. H. J. & Waterhouse, P. M. RNAi for insect-proof plants. Nat. Biotechnol. 25, 1231–1232 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Lilley, C. J., Davies, L. J. & Urwin, P. E. RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139, 630–640 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Koch, A. et al. Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species. Proc. Natl Acad. Sci. USA 110, 19324–19329 (2013).

    CAS  Article  Google Scholar 

  11. 11

    Duan, C.-G., Wang, C.-H. & Guo, H.-S. Application of RNA silencing to plant disease resistance. Silence 3, 5 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Robinson, K. E., Worrall, E. A. & Mitter, N. Double stranded RNA expression and its topical application for non-transgenic resistance to plant viruses. J. Plant Biochem. Biotechnol. 23, 231–237 (2014).

    CAS  Article  Google Scholar 

  13. 13

    Tenllado, F., Llave, C. & Diaz-Ruiz, J. R. RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Res. 102, 85–96 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Tenllado, F. & Díaz-Ruíz, J. R. Double-stranded RNA-mediated interference with plant virus infection. J. Virol. 75, 12288–12297 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Tenllado, F., Martínez-García, B., Vargas, M. & Díaz-Ruíz, J. R. Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol. 3, 3 (2003).

    Article  Google Scholar 

  16. 16

    Gan, D. et al. Bacterially expressed dsRNA protects maize against SCMV infection. Plant Cell Rep. 29, 1261–1268 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Lau, S. E. et al. Crude extracts of bacterially-expressed dsRNA protect orchid plants against Cymbidium mosaic virus during transplantation from in vitro culture. J. Hortic. Sci. Biotechnol. 89, 569–576 (2014).

    Article  Google Scholar 

  18. 18

    Xu, Z. P. et al. Stable suspension of layered double hydroxide nanoparticles in aqueous solution. J. Am. Chem. Soc. 128, 36–37 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Ram Reddy, M. K., Xu, Z. P., Lu, G. Q. & Diniz da Costa, J. C. Layered double hydroxides for CO2 capture: structure evolution and regeneration. Ind. Eng. Chem. Res. 45, 7504–7509 (2006).

    CAS  Article  Google Scholar 

  20. 20

    Ram Reddy, M. K., Xu, Z. P., Lu, G. Q. (Max) & Diniz da Costa, J. C. Effect of SOx adsorption on layered double hydroxides for CO2 capture. Ind. Eng. Chem. Res. 47, 7357–7360 (2008).

    Article  Google Scholar 

  21. 21

    Xu, Z. & Zeng, H. Abrupt structural transformation in hydrotalcite-like compounds Mg1-xAlx(OH)2(NO3)x.nH2O as a continuous function of nitrate anions. J. Phys. Chem. B 105, 1743–1749 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Dong, H. et al. Engineering small MgAl-layered double hydroxide nanoparticles for enhanced gene delivery. Appl. Clay Sci. 100, 66–75 (2014).

    CAS  Article  Google Scholar 

  23. 23

    Cavani, F., Trifirò, F. & Vaccari, A. Hydrotalcite-type anionic clays: preparation, properties and applications. Catalysis Today 11, 173–301 (1991).

    CAS  Article  Google Scholar 

  24. 24

    Dietzgen, R. & Mitter, N. Transgenic gene silencing strategies for virus control. Australas. Plant Pathol. 35, 605–618 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Li, H.-W. et al. Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism. EMBO J. 18, 2683–2691 (1999).

    CAS  Article  Google Scholar 

  26. 26

    Ladewig, K., Niebert, M., Xu, Z. P., Gray, P. P. & Lu, G. Q. M. Efficient siRNA delivery to mammalian cells using layered double hydroxide nanoparticles. Biomaterials 31, 1821–1829 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Elmayan, T. et al. Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747–1757 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Roossinck, M. J. Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses. J. Virol. 76, 3382–3387 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).

    CAS  Article  Google Scholar 

  30. 30

    Wittrup, A. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33, 870–876 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Jiang, L. et al. Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA. Nanoscale 6, 9965–9969 (2014).

    CAS  Article  Google Scholar 

  32. 32

    Ladewig, K., Xu, Z. P. & Lu, G. Q. (Max). Layered double hydroxide nanoparticles in gene and drug delivery. Expert Opin. Drug Deliv. 6, 907–922 (2009).

    CAS  Article  Google Scholar 

  33. 33

    Li, H., Guan, R., Guo, H. & Miao, X. New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests. Plant Cell Environ. 38, 2277–2285 (2015).

    CAS  Article  Google Scholar 

  34. 34

    Lau, S.-E., Schwarzacher, T., Othman, R. Y. & Harikrishna, J. A. dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid. BMC Plant Biol. 15, 194 (2015).

    Article  Google Scholar 

  35. 35

    Molnar, A. et al. Plant virus-derived small interfering RNAs originate predominantly from high structured single-strand viral RNAs. J. Virol. 79, 7812–7818 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Donaire, L. et al. Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 392, 203–214 (2009).

    CAS  Article  Google Scholar 

  37. 37

    Mitter, N., Koundal, V., Williams, S. & Pappu, H. Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants. PLoS ONE 8, e76276 (2013).

    CAS  Article  Google Scholar 

  38. 38

    Mitter, N. & Dietzgen, R. G. Use of hairpin RNA constructs for engineering plant virus resistance. Methods Mol. Biol. 894, 191–208 (2012).

    CAS  Article  Google Scholar 

  39. 39

    Mitter, N., Sulistyowati, E. & Dietzgen, R. G. Cucumber mosaic virus infection transiently breaks dsRNA-induced transgenic immunity to Potato virus Y in tobacco. Am. Phytopathological Soc. 16, 936–944 (2003).

    CAS  Google Scholar 

  40. 40

    Sulistyowati, E., Mitter, N., Bastiaan-Net, S., Roossinck, M. J. & Dietzgen, R. G. Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus. Australas. Plant Pathol. 33, 505–512 (2004).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing