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Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids

Nature Biotechnology volume 32, pages 102–105 (2014)Cite this article

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Abstract

The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum, without virion assembly. We show that when four aphid pest species—A. pisum, Rhopalosiphum padi, Aphis glycines and Myzus persicae—feed on a recombinant coat protein–toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein–insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

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Figure 1: Constructs used to test for PEMV CP-P–mediated delivery of GFP and Hv1a to aphids.
Figure 2: Mortality of four species of aphid fed on CP-P-Hv1a or control treatments (CP-P-Hv1am, Hv1a or mock) in membrane feeding assays.
Figure 3: Aphid populations on transgenic Arabidopsis.

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References

  1. Sanahuja, G., Banakar, R., Twyman, R.M., Capell, T. & Christou, P. Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J. 9, 283–300 (2011).

    Article  CAS  Google Scholar 

  2. Stokstad, E. & Grullon, G. Infographic: pesticide planet. Science 341, 730–731 (2013).

    Article  Google Scholar 

  3. Chougule, N.P. & Bonning, B.C. Toxins for transgenic resistance to hemipteran pests. Toxins 4, 405–429 (2012).

    Article  CAS  Google Scholar 

  4. Li, H., Chougule, N.P. & Bonning, B.C. Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris). J. Invertebr. Pathol. 107, 69–78 (2011).

    Article  CAS  Google Scholar 

  5. Lu, Y. et al. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science 328, 1151–1154 (2010).

    Article  CAS  Google Scholar 

  6. Li, G. et al. Effects of transgenic Bt cotton on the population density, oviposition behavior, development, and reproduction of a nontarget pest, Adelphocoris suturalis (Hemiptera: Miridae). Environ. Entomol. 39, 1378–1387 (2010).

    Article  Google Scholar 

  7. Schmidt, N.R. & Bonning, B.C. in Molecular Biology and Genetics of Lepidoptera (eds. M. Goldsmith & F. Marc) 307–319 (Taylor and Francis, Baton Rouge, FL, 2010).

  8. King, G.F. & Hardy, M.C. Spider-venom peptides: structure, pharmacology, and potential for control of insect pests. Annu. Rev. Entomol. 58, 475–496 (2013).

    Article  CAS  Google Scholar 

  9. Reavy, B. & Mayo, M.A. Persistent transmission of luteoviruses by aphids. Adv. Bot. Res. 36, 21–46 (2002).

    Article  CAS  Google Scholar 

  10. Gray, S. & Gildow, F.E. Luteovirus-aphid interactions. Annu. Rev. Phytopathol. 41, 539–566 (2003).

    Article  CAS  Google Scholar 

  11. Liu, S., Sivakumar, S., Wang, Z., Bonning, B.C. & Miller, W.A. The readthrough domain of Pea enation mosaic virus coat protein is not essential for virus stability in the hemolymph of the pea aphid. Arch. Virol. 154, 469–479 (2009).

    Article  CAS  Google Scholar 

  12. Fletcher, J.I. et al. The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nat. Struct. Biol. 4, 559–566 (1997).

    Article  CAS  Google Scholar 

  13. Pal, N., Yamamoto, T., King, G.F., Waine, C. & Bonning, B.C. Aphicidal efficacy of scorpion- and spider-derived neurotoxins. Toxicon 70, 114–122 (2013).

    Article  CAS  Google Scholar 

  14. Sivakumar, S. et al. Baculovirus-expressed virus-like particles of Pea enation mosaic virus vary in size and encapsidate baculovirus mRNAs. Virus Res. 139, 54–63 (2009).

    Article  CAS  Google Scholar 

  15. Lamb, J.W. et al. Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus. J. Gen. Virol. 77, 1349–1358 (1996).

    Article  CAS  Google Scholar 

  16. Locke, M. & Russell, V.W. in Microscopic Anatomy of Invertebrates, vol. 11B (eds. F.W. Harrison & M. Locke) 687–709 (Wiley-Liss Inc., New York, 1998).

  17. Crossley, A.C. in Comprehensive Insect Physiology, Biochemistry, and Pharmacology (eds. G.A. Kerkut & L.I. Gilbert) 487–515 (Pergamon Press, New York, 1985).

  18. Wang, X. et al. Structure-function studies of ω-atracotoxin, a potent antagonist of insect voltage-gated calcium channels. Eur. J. Biochem. 264, 488–494 (1999).

    Article  CAS  Google Scholar 

  19. Tedford, H.W., Fletcher, J.I. & King, G.F. Functional significance of the beta hairpin in the insecticidal neurotoxin ω-atracotoxin-Hv1a. J. Biol. Chem. 276, 26568–26576 (2001).

    Article  CAS  Google Scholar 

  20. Jeffers, L.A. & Michael Roe, R. The movement of proteins across the insect and tick digestive system. J. Insect Physiol. 54, 319–332 (2008).

    Article  CAS  Google Scholar 

  21. Maiti, I.B., Oey, N., Dahlman, D.L. & Webb, B.A. Antibiosis-type resistance in transgenic plants expressing a teratocyte secretory peptide (TSP) gene from a hymenopteran endoparasite (Microplitis croceipes). Plant Biotechnol. J. 1, 209–219 (2003).

    Article  CAS  Google Scholar 

  22. Tortiglione, C. et al. The expression in tobacco plants of Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF) alters growth and development of the tobacco budworm, Heliothis virescens. Mol. Breed. 9, 159–169 (2002).

    Article  CAS  Google Scholar 

  23. Gray, S.M. & Banerjee, N. Mechanisms of arthropod transmission of plant and animal viruses. Microbiol. Mol. Biol. Rev. 63, 128–148 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Fitches, E.C., Pyati, P., King, G.F. & Gatehouse, J.A. Fusion to snowdrop lectin magnifies the oral activity of insecticidal ω-hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS ONE 7, e39389 (2012).

    Article  CAS  Google Scholar 

  25. Fitches, E.C. et al. Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders. Pest Manag. Sci. 66, 74–83 (2010).

    Article  CAS  Google Scholar 

  26. Bonning, B.C. & Chougule, N.P. Delivery of intrahemocoelic peptides for insect pest management. Trends Biotechnol. (in the press) (2013).

  27. Pennacchio, F., Giordana, B. & Rao, R. in Parasitoid Viruses: Symbionts and Pathogens (eds. N.E. Beckage & J.M. Drezen) 269–283 (Academic Press, San Diego, 2012).

  28. Demler, S.A., Rucker-Feeney, D.G., Skaf, J.S. & deZoeten, G.A. Expression and suppression of circulative aphid transmission in pea enation mosaic virus. J. Gen. Virol. 78, 511–523 (1997).

    Article  CAS  Google Scholar 

  29. Fukatsu, T., Tsuchida, T., Nikoh, N. & Koga, R. Spiroplasma symbiont of the pea aphid, Acyrthosiphon pisum (Insecta: Homoptera). Appl. Environ. Microbiol. 67, 1284–1291 (2001).

    Article  CAS  Google Scholar 

  30. Tedford, H.W. et al. Scanning mutagenesis of ω-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels. J. Biol. Chem. 279, 44133–44140 (2004).

    Article  CAS  Google Scholar 

  31. Febvay, G., Delobel, B. & Rahbe, Y. Influence of the amino-acid balance on the improvement of an artificial diet for a biotype of Acyrthosiphon pisum (Homoptera, Aphididae). Can. J. Zool. 66, 2449–2453 (1988).

    Article  CAS  Google Scholar 

  32. Liu, S., Bonning, B.C. & Miller, W.A. A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel. J. Virol. Methods 132, 174–180 (2006).

    Article  CAS  Google Scholar 

  33. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  34. Collins, T.J. ImageJ for microscopy. Biotechniques 43, S25–S30 (2007).

    Article  Google Scholar 

  35. Hughes, P.R., Van Beek, N.A.M. & Wood, H.A. A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae. J. Invertebr. Pathol. 48, 187–192 (1986).

    Article  Google Scholar 

  36. SAS Institute. SAS/STAT(R) 9.3 User's Guide (SAS Institute Inc., Cary NC, 2013).

  37. LeOra-Software POLO-PC. A User's Guide to Probit and Logit Analysis (LeOra Software, Berkeley, California, 1987).

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Acknowledgements

We thank R. Harrison, US Department of Agriculture (USDA), Beltsville, Maryland, for construction of the baculovirus expressing CP-GFP and for production of the PEMV coat protein antiserum. This work was supported by the USDA North Central Biotechnology Initiative grant number 97-34340-3987, USDA National Research Initiative grant number 02-01280, Australian Research Council Discovery Grant DP0774245, Bayer Crop Science, Syngenta, DuPont Pioneer, Grow Iowa Values Funds, Iowa State University Plant Sciences Institute, and by Hatch Act and State of Iowa funds. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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Authors and Affiliations

  1. Department of Entomology, Iowa State University, Ames, Iowa, USA

    Bryony C Bonning, Narinder Pal, Sijun Liu & S Sivakumar

  2. Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa, USA

    Zhaohui Wang & W Allen Miller

  3. Department of Statistics, Iowa State University, Ames, Iowa, USA

    Philip M Dixon

  4. Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia

    Glenn F King

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  1. Bryony C Bonning
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Contributions

W.A.M. devised the coat protein–toxin fusion strategy; B.C.B. and W.A.M. planned the experiments, interpreted results and wrote the manuscript; N.P. conducted E. coli expression of fusion proteins, generation and molecular testing of transgenic plants, membrane feeding assays and aphid feeding bioassays with transgenic plants; S.L. conducted aphid microinjections and preliminary experiments; Z.W. constructed plasmids and performed preliminary experiments; S.S. conducted research with baculovirus expressed fusions; G.F.K. provided oversight for use of the Hv1a toxin. P.M.D. conducted the statistical analyses.

Corresponding authors

Correspondence to Bryony C Bonning or W Allen Miller.

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Competing interests

The coat protein-toxin fusion strategy has been patented by B.C.B. and W.A.M.: US Patent No. 7,312,080 (2007). "Plant resistance to insect pests mediated by viral proteins." G.F.K. is a member of the Scientific Advisory Board of Vestaron Corporation, a company that is developing spider peptides as insecticides.

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Bonning, B., Pal, N., Liu, S. et al. Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids. Nat Biotechnol 32, 102–105 (2014). https://doi.org/10.1038/nbt.2753

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