Pest and disease protection conferred by expression of barley β-hordothionin and Nicotiana alata proteinase inhibitor genes in transgenic tobacco
Julia A. Charity A E , Peter Hughes A , Marilyn A. Anderson B , Dennis J. Bittisnich C , Malcolm Whitecross D and T. J. V. Higgins AA CSIRO, Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia.
B Department of Biochemistry and Genetics, La Trobe University, Bundoora, Vic. 3083, Australia.
C CRC for Plant Science, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia.
D Department of Botany and Zoology, Australian National University, Canberra, ACT 2601, Australia.
E Present address: Forest Research, Private Bag 3020, Rotorua, New Zealand. Corresponding author. Email: julia.charity@forestresearch.co.nz
Functional Plant Biology 32(1) 35-44 https://doi.org/10.1071/FP04105
Submitted: 11 June 2004 Accepted: 11 November 2004 Published: 21 January 2005
Abstract
Proteinase inhibitors and thionins are among the many proteins thought to have a role in plant defence against pests and pathogens. Complementary DNA clones encoding the precursors of a multi-domain proteinase inhibitor from Nicotiana alata Link et Otto (NA-PI) (Mr approximately 43 000) and a β-hordothionin (β-HTH) (Mr approximately 13 000) from barley, were linked to constitutive promoters and subsequently transferred by Agrobacterium-mediated transformation into tobacco. The NA-PI and β-HTH precursor proteins were synthesised and post-translationally processed in transgenic tobacco and accumulated as polypeptides of apparent size Mr approximately 6000 and Mr approximately 8500, respectively. The na-pi and β-hth genes were stably inherited for at least two generations. Transgenic tobacco plants containing the highest amounts of NA-PI and β-HTH were crossed to produce plants containing both genes. Helicoverpa armigera (tobacco budworm) larvae that ingested transgenic tobacco leaves expressing both NA-PI and β-HTH, exhibited higher mortality and slower development relative to larvae fed on non-transgenic tobacco. NA-PI and β-HTH, either alone, or in combination, also conferred protection against the fungal pathogen, Botrytis cinerea (grey mould) and the bacterial pathogen, Pseudomonas solanacearum (bacterial wilt). The effect of the two proteins depended upon the organism tested and the contribution of each gene to the protective effects was not necessarily equal. The genetic engineering of plants with proteinase inhibitors or thionins, therefore, has potential for improving crop productivity by simultaneously increasing resistance to both pests and pathogens.
Keywords: Botrytis cinerea, defence genes, Helicoverpa armigera, insect-, bacterial- and fungal-resistance, Pseudomonas syringae.
Acknowledgments
JAC was supported by an Australian National University PhD scholarship and by funding from the CRC for Plant Science. PH was supported by a PhD Scholarship from Cotton Seed Distributors. We thank Melbourne University for supplying the na-pi gene sequence used in pAM8 and Dr Marcus Lee for sequencing the construct. We acknowledge Dr Ross Cunningham, Dr Christine Donnelly and Dr Roger Morton for assistance with statistical analysis and Mrs Anna Grabowski for rearing neonates for this study. We also thank Professor Richard Gardner, Dr Derek White, Dr Danny Llewellyn, Professor Don Spencer and the two anonymous reviewers for critical review of the manuscript before publication. Mrs Tania Elder and Mrs Bev Harniss are acknowledged for preparing the manuscript for publication.
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