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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Flood, J. The importance of plant health to food security. Food Secur. 2, 215–231 (2010).
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).
Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).
Bartel, D. P. MicroRNAs: genomics, biogensis, mechanism and function. Cell 116, 281–297 (2004).
Burand, J. P. & Hunter, W. B. RNAi: future in insect management. J. Invertebr. Pathol. 112, S68–S74 (2013).
Mailard, P. V. et al. Antiviral RNA interference in mammalian cells. Science 342, 235–238 (2013).
Borges, F. & Martienssen, R. A. The expanding world of small RNAs in plants. Nat. Rev. Mol. Cell Biol. 16, 727–741 (2015).
Gordon, K. H. J. & Waterhouse, P. M. RNAi for insect-proof plants. Nat. Biotechnol. 25, 1231–1232 (2007).
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).
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).
Duan, C.-G., Wang, C.-H. & Guo, H.-S. Application of RNA silencing to plant disease resistance. Silence 3, 5 (2012).
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).
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).
Tenllado, F. & Díaz-Ruíz, J. R. Double-stranded RNA-mediated interference with plant virus infection. J. Virol. 75, 12288–12297 (2001).
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).
Gan, D. et al. Bacterially expressed dsRNA protects maize against SCMV infection. Plant Cell Rep. 29, 1261–1268 (2010).
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).
Xu, Z. P. et al. Stable suspension of layered double hydroxide nanoparticles in aqueous solution. J. Am. Chem. Soc. 128, 36–37 (2006).
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).
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).
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).
Dong, H. et al. Engineering small MgAl-layered double hydroxide nanoparticles for enhanced gene delivery. Appl. Clay Sci. 100, 66–75 (2014).
Cavani, F., Trifirò, F. & Vaccari, A. Hydrotalcite-type anionic clays: preparation, properties and applications. Catalysis Today 11, 173–301 (1991).
Dietzgen, R. & Mitter, N. Transgenic gene silencing strategies for virus control. Australas. Plant Pathol. 35, 605–618 (2006).
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).
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).
Elmayan, T. et al. Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747–1757 (1998).
Roossinck, M. J. Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses. J. Virol. 76, 3382–3387 (2002).
Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).
Wittrup, A. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33, 870–876 (2015).
Jiang, L. et al. Systemic gene silencing in plants triggered by fluorescent nanoparticle-delivered double-stranded RNA. Nanoscale 6, 9965–9969 (2014).
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).
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).
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).
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).
Donaire, L. et al. Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 392, 203–214 (2009).
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).
Mitter, N. & Dietzgen, R. G. Use of hairpin RNA constructs for engineering plant virus resistance. Methods Mol. Biol. 894, 191–208 (2012).
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).
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).
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.
The authors declare no competing financial interests.
About this article
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
Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs
Applied Microbiology and Biotechnology (2020)
Journal of Agricultural and Food Chemistry (2020)
DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya
Journal of Virological Methods (2020)
Applied Microbiology and Biotechnology (2020)
Analysis of RNA Interference (RNAi) Biopesticides: Double-Stranded RNA (dsRNA) Extraction from Agricultural Soils and Quantification by RT-qPCR
Environmental Science & Technology (2020)