PYATI, PRASHANT,SHIVASHARAN (2010) Molecular approaches to increasing resistance of wheat (Triticum aestivum L.) towards two insect pests; Cereal aphid (Sitobion avenae F.) and Wheat bulb fly (Delia coarctata Fallen). Doctoral thesis, Durham University.
Cereal aphid (Sitobion avenae) and wheat bulb fly (Delia coarctata) are serious pests of wheat in the UK. At the present, chemical pesticides are used to control these insects, but they are limited in effectiveness, and have undersirable ecological impacts. There is a need to improve wheat genetically to be resistant to such inset pests. The objectives of this work were to investigate digestive biochemistry in the selected insect pests of wheat, and to determine effects of potential endogenous resistance factors in wheat on digestion, nutrition and other insect metabolic processes. The aim was to develop new strategies for crop protection.
Digestive biochemistry in S. avenae and D. coarctata was studied to characterise gut proteases and their inhibition by host plant proteinase inhibitors (PIs). Investigation of proteolytic digestion in S. avenae gut showed that in spite of being a phloem-feeding insect, cereal aphid could digest ingested protein, using cysteine proteases. D. coarctata larvae contained mainly serine protease activity. A serine protease (DcSP) and a cysteine protease (DcCathL) from D. coarctata gut tissue were expressed as recombinant proteins. Only DcCathL was recovered in active form. DcCathL was insecticidal to Mamestra brassicae when injected into hemolymph, causing systemic and extensive melanisation. DcCathL selectively degraded recombinant serpins from M. brassicae in in vitro assays, and is suggested to interfere with regulation of the proteolytic cascade leading to phenoloxidase activation and melanin production in vivo. DcCathL has potential as a biopesticide if it could be made effective when orally delivered. A cationic amino acid transporter from D. coarctata gut (DcCAAT) was also cloned as a target for RNA interference.
Potential resistance factors in wheat were characterised by expression as recombinant proteins. Two PIs from wheat (subtilisin/chymotrypsin inhibitor; WSCI, and cysteine proteinase inhibitor; WCPI) were expressed in the yeast Pichia pastoris, and purified. WSCI inhibited gut protease activity of both insects in in vitro and in vivo assays, whereas WCPI only inhibited S. avenae gut extract activity. On feeding, WSCI was antimetabolic to both insects, affecting both survival and growth, whereas WCPI was antimetabolic to S. avenae only. Wheat Hessian fly responsive (Hfr) genes are up-regulated in response to herbivory by Hessian fly (Mayetiola destructor). The protein product Hfr-3 was expressed and purified, and showed antimetabolic effects on survival and growth of both S. avenae and D. coarctata. Both accumulated and induced defence proteins, like WSCI, WCPI and Hfr-3, have the potential to act as endogenous resistance factors in wheat towards a range of insect pests. Developing a wheat variety constitutively expressing these defence proteins by using traditional breeding methods and/or modern biotechnological tools is discussed.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Delia coarctata. Sitobion avenae. Hessian fly, Hfr, Pichia|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||06 Jul 2010 16:27|