Skip to main content
SpringerLink
Log in

Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco

  • Published:
Molecular Breeding Aims and scope Submit manuscript

Abstract

A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T0 and T1 plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. An G, Watson BD, Stachel S, Gordon MP, Nester EW: New cloning vehicles for transformation of higher plants. EMBO J 4: 277–284 (1985).

    Google Scholar 

  2. Argall ME, Bradbury JH, Shaw DC: Amino-acid sequence of a trypsin/chymotrypsin inhibitor from giant taro (Alocasia macrorrhiza). Biochim Biophys Acta 1204: 189–194 (1994).

    Google Scholar 

  3. Bolter CJ, Jongsma MA: Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 41: 1071–1078.

  4. Boulter D, Edwards GA, Gatehouse AMR, Gatehouse JA, Hilder VA: Additive protective effects of incorporating two different higher plant derived insect resistance genes in transgenic tobacco plants. Crop Prot 9: 351–354 (1990).

    Google Scholar 

  5. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  6. Broadway RM: Are insects resistant to plant proteinase inhibitors? J Insect Physiol 41: 107–116 (1995).

    Google Scholar 

  7. Broadway RM: Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 32: 39–53 (1996).

    Google Scholar 

  8. Broadway RM, Duffy SS: Plant Proteinase Inhibitors: mechanism of action and effects on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 32: 827–833 (1986).

    Google Scholar 

  9. Burgess EPJ, Stevens PS, Keen GK, Laing WA, Christeller LT: Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Entomol Exp Appl 61: 123–130 (1991).

    Google Scholar 

  10. Christeller JT, Shaw BD, Gardiner SE, Dymock J: Partial purification and characterisation of the major midgut proteases of grass grub larvae (Costelytra zealandica; Coleoptera: Scarabaeidae). Insect Biochem 19: 221–231 (1989).

    Google Scholar 

  11. Christeller JT, Laing WA, Shaw BD, Burgess EPJ: Characterisation and partial purification of the digestive proteases of the black field cricket, Teleogryllus commodus (Walker): Elastase is a major component. Insect Biochem 20: 157–164 (1990).

    Google Scholar 

  12. Christeller JT, Laing WA, Markwick NP, Burgess EPJ: Midgut protease activities in 12 phytophagous lepidopteran larvae: dietary and protease inhibitor interactions. Insect Biochem Mol Biol 22: 735–746 (1992).

    Google Scholar 

  13. De Almeida ERP, Gossele V, Muller CG, Dockx J, Reynaerts A, Botterman J, Krebbers E, Timko MP: Transgenic expression of twomarker genes under the control of an Arabidopsis rbcS promoter: sequences encoding the Rubisco transit peptide increase expression level. Mol Gen Genet 218: 78–86 (1989).

    Google Scholar 

  14. Ditta G, Stanfield S, Corbin D, Helsinki DR: Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77: 7347–7351 (1980).

    Google Scholar 

  15. Duan X, Li X, Xue Q, Abo-El-Saad, Xu D, Wu R: Transgenic rice plants harbouring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnol 14: 494–498 (1996).

    Google Scholar 

  16. Ellis JG, Llewellyn DJ, Dennis ES, Peacock WJ: Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco EMBO J 6: 11–16 (1987).

    Google Scholar 

  17. Fischhoff DA, Bowdish KS, Perlak FJ, Marrone PG, McCormick SM, Niedermeyer JG, Dean DA, Kusano-Kretzmer K, Mayer EJ, Rochester DE, Rogers SG, Fraley RT: Insect tolerant transgenic tomato plants. Bio/technology 5: 807–813 (1987).

    Google Scholar 

  18. Gatehouse AMR, Hilder VA: Genetic manipulation of crops for insect resistance. In: Marshall G, Walters D (eds) Molecular Biology in Crop Protection, pp. 176–201. Chapman and Hall, London (1994).

    Google Scholar 

  19. Green TR, Ryan CA: Wound-induced proteinase inhibitor in plant leaves: a possible defence mechanism against insects. Science 175: 776–777 (1972).

    Google Scholar 

  20. Green TR, Ryan CA: Wound-induced proteinase inhibitor in tomato leaves. Plant Physiol 51: 19–21 (1973).

    Google Scholar 

  21. Hammer BC, Shaw DC, Bradbury JH: Trypsin inhibitor from Colocasia esculenta, Alocasia macrorrhiza and Cyrtosperma chamissonis. Phytochemistry 28: 3019–3026 (1989).

    Google Scholar 

  22. Herman EM, Tague BW, Hoffman LM, Kjemtrup SE, Chrispeels MJ: Retention of phytohemagglutinin with carboxyterminal tetrapeptide in the nuclear envelope and the endoplasmic reticulum. Planta 182: 305–312 (1990).

    Google Scholar 

  23. Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D: A novel mechanism of insect resistance engineered into tobacco. Nature 300: 160–163 (1987).

    Google Scholar 

  24. Hummel BCW: A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Can J Biochem Physiol 37: 1393–1399 (1959).

    Google Scholar 

  25. Johnson R, Narvaez J, An G, Ryan C: Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defence against Manduca sexta. Proc Natl Acad Sci USA 86: 9871–9875 (1989).

    Google Scholar 

  26. Johnston KA, Lee MJ, Gatehouse JA, Anstee JH: The partial purification and characterisation of serine protease activity in midgut of larval Helicoverpa armigera. Insect Biochem 21: 389–397 (1991).

    Google Scholar 

  27. Johnston KA, Gatehouse JA, Anstee JH: Effects of soybean protease inhibitor on the growth and development of larval Helicoverpa armigera. J Insect Physiol 39: 657–664 (1993).

    Google Scholar 

  28. Jongsma MA, Bakker PL, Peters J, Bosch D, Stiekema WJ: Adaptation of Spodoptera exigua larvae to plant proteinase inhibitor by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 92: 8041–8045 (1995).

    Google Scholar 

  29. Jongsma MA, Stiekema WJ, Bosch D: Combating inhibitorinsensitive proteases of insect pests. Trends Biotechnol 14: 331–333 (1996).

    Google Scholar 

  30. Landsmann J, Llewellyn DJ, Dennis ES, Peacock WJ: Organ regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants. Mol Gen Genet 214: 68–73 (1988).

    Google Scholar 

  31. Leplé JC, Bonadé-Bottino M, Augustin S, Pilate G, Dumanois Lê Têan V, Delplanque A, Cornu D, Jouanin L: Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breed 1: 319–328 (1995).

    Google Scholar 

  32. McPherson S, Perlak FJ, Fuchs RL, MacIntosh SC, Dean DA, Fischoff DA: Expression and analysis of the insect control protein from Bacillus thuringiensis var. tenebrionis. In: Abstracts of the First International Symposium on the Molecular Biology of the Potato, Bar Harbor, ME, p. 51 (1989).

  33. Mathews A, Llewellyn DJ, Wu Y, Dennis ES: Isolation and characterisation of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor. Plant Mol Biol 30: 1035–1039 (1996).

    Google Scholar 

  34. Metcalf RL: The ecology of insecticides and the chemical control of insects. In: Kogan M (ed) Ecological Theory and Integrated Pest Management, pp. 251–297. John Wiley, New York (1986).

    Google Scholar 

  35. Munro S, Pelham HRB: AC-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907 (1987).

    Google Scholar 

  36. Pelham HRB: The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci 15: 483–486 (1990).

    Google Scholar 

  37. Peng L, Bradbury JH, Hammer BC, Shaw DC: Comparison of amino acid sequences of the trypsin inhibitors from taro (Colocasia esculenta), giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis). Biochem and Mol Biol Int 31: 73–81 (1993).

    Google Scholar 

  38. Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischoff DA: Insect resistant cotton plants. Bio/technology 8: 3324–3328 (1990).

    Google Scholar 

  39. Purcell JP, Greenplate JT, Sammons RD: Examination of midgut luminal protease activities in six economically important insects. Insect Biochem Mol Biol 22: 41–47 (1992).

    Google Scholar 

  40. Pyke B: Pupae control can cut costs and reduce resistance. Aust Cotton Grower 16: 15–16 (1995).

    Google Scholar 

  41. Ryan CA: Protease Inhibitors in Plants: Genes for improving defences against insects and pathogens. Annu Rev Phytopath 28: 425–449 (1990).

    Google Scholar 

  42. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  43. Shade RE, Schroeder HE, Pueyo JJ, Tabe LM, Murdock LL, Higgins TJV, Chrispeel MJ: Transgenic pea seeds expressing the _-amylase inhibitor of the common bean are resistant to bruchid beetles. Bio/technology 12: 793–796 (1994).

    Google Scholar 

  44. Spencer D, Higgins TJV, Button SC, Davey RA: Pulselabelling studies on protein synthesis in developing pea seeds and evidence of a precursor form of legumin small subunit. Plant Physiol 66: 510–520 (1980).

    Google Scholar 

  45. Tabe LM, Wardley-Richardson T, Ceriotti A, Aryan A, McNabb W, Moore A, Higgins TJV: A biotechnological approach to improving the nutritive value of alfalfa. J Anim Sci 73: 2752–2759 (1995).

    Google Scholar 

  46. Vaeck M, Reynaerts A, Hofte H, Jansens S, De Beuckeleer M, Dean C, Zabeau M, Van Montagu M, Leemans J: Transgenic plants protected from insect attack. Nature 327: 33–37 (1987).

    Google Scholar 

  47. Walsh KA, Wilcox PE: Serine proteases. In: Perlmann GE, Lorand L (eds) Methods in Enzymology, vol. 19. Proteolytic Proteolytic Enzymes, pp. 000–000. Academic Press, NY and London (1970).

    Google Scholar 

  48. Wendelt CI, Khan MRI, Craig S, Schhroeder HE, Spencer D, Higgins TJV: Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J 2: 181–192 (1992).

    Google Scholar 

  49. Xu D, Xue Q, McElroy D, Mawal Y, Hilder VA, Wu R: Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice confers resistance to two major rice insect pests. Mol Breed 2: 167–173 (1996).

    Google Scholar 

Download references

Author information

Author notes

  1. Anne Mathews

    Present address: Faculty of Agriculture, University of Western Australia, Nedlands, 6009, Australia

Authors and Affiliations

  1. CRC for Sustainable Cotton Production, CSIRO Plant Industry, P.O. Box 1600, Canberra City, ACT, 2601, Australia

    Yingru Wu, Danny Llewellyn & Elizabeth S. Dennis

Authors
  1. Yingru Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Danny Llewellyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne Mathews
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elizabeth S. Dennis
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, Y., Llewellyn, D., Mathews, A. et al. Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Molecular Breeding 3, 371–380 (1997). https://doi.org/10.1023/A:1009681323131

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1009681323131

Search

Navigation