Durham E-Theses Alimentary tract proteinases of the Southern corn rootworm (Diabrotica undecimpunctata howardi) and the potential of potato Kunitz proteinase inhibitors for larval control. Macgregor, James Mylne How to cite: Macgregor, James Mylne (2001) Alimentary tract proteinases of the Southern corn rootworm (Diabrotica undecimpunctata howardi) and the potential of potato Kunitz proteinase inhibitors for larval control., Durham theses, Durham University. 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The copyright of this thesis rests with the author. No quotation from it should be published without his prior written consent and information derived from it should be acknowledged. A thesis submitted by James Mylne Macgregor B.Sc. in accordance with the requirements of the University ofDurham for the degree ofDoctor of Philosophy. Department ofBiological Sciences, University of Durham. September 200 I. Abstract. Proteolytic digestion by larval Diabrotica undecimpunctata howardi (Barber) (D. undecimpunctata) has been investigated with the aim ofproducing transgenic plants possessing enhanced resistance to this economically important crop pest. Biochemical characterisation in vitro by pH dependent hydrolysis and inhibition assays incorporating E-64, pepstatin A and soybean Kunitz trypsin inhibitor showed the majority of hydrolytic activity occurs at pH 5.5 and is performed by cysteine and aspartic endopeptidases. Cysteine and aspartic proteinase encoding clones were isolated from a larval alimentary tract cDNA library. Four cathepsin L-like cysteine proteinases and two cathepsin D-like aspartic proteinase cDNA clones were identified by coding homology to known proteinase sequences. Analysis of primary and secondary sequence features revealed D. undecimpunctata aspartic proteinase 1 exhibits features associated with cathepsins E and is proposed to be a D. undecimpunctata cathepsin E-like aspartic proteinase. Cathepsin D-like aspartic proteinase inhibitors ofthe potato Kunitz proteinase inhibitor (PKPI) family have been isolated by PCR and expressed employing the pET expression system (Novagen). In vitro assays demonstrated the inhibitory activity of PKPI-A and PKPI-B inhibitors against larval D. undecimpunctata alimentary tract proteolytic enzymes. To the authors knowledge this work represents the first reporting of the expression and purification ofbiologically active PKPI proteins. In vitro assays incorporating oryzacystatin I and PKPI proteins resulted in increased inhibition of proteolytic activity compared to single inhibitor and uninhibited control reactions. Inhibition assays provide evidence for the potential ofadual proteinase inhibitor strategy to arrest protein hydrolysis by larval D. undecimpunctata, preventing essential amino acid absorption. Further research is necessary to characterise the properties ofthe digestive enzymes isolated in this work and the inhibitory spectrum ofPKPI proteins. Transgenic crops expressing a combination of oryzacystatin and PKPI proteins would be predicted to show enhanced resistance to insect herbivores by virtue of digestive proteolysis inhibition. Acknowledgements. I would like to express my appreciation for the financial support provided by the BBSRC and Syngenta PLC for this C.A.S.E studentship. My gratitude is also due to the Department of Biological Sciences, Durham University for the use of their facilities. My appreciation is extended to Dr John Gatehouse and Dr Jane Cayley for assistance and supervision during the pursuit of this project. My appreciation also goes to Gillian Davison for maintaining the D. undecimpunctata cultures and for being flexible with changing plans. Many thanks go to the following people; Ilona Bausch. Samantha Connors. Phi lip Pattison and Jaqueline Snowden Terry Pratchett for his obnoxious but accurate little phrase. Ana Gabriella Renetario Rojas. Vincent Townshend and Lynne Conlon. Also to Florence Reeves, Jack O'Grady and Colin Illet, who are sadly no longer with us. To my parents possibly the greatest thanks for their advice and support. II a. a. amino acid. bp base pair. cDNA copy deoxyribonucleic acid. D. undecimpunctata Diabrotica undecimpunctata howardi Barber. DNA deoxyribonucleic acid. EPPO European and Mediterranean Plant Protection Organisation. g gram. hr hour. L litre. M Molar. mm minute. MMLV-RT­ Moloney Murine Leukemia Virus reverse transcriptase. mRNA messenger ribonucleic acid. MCS multiple cloning site. O.D. optical density. PAGE polyacrylamide gel electrophoresis. PCR polymerase chain reaction. pfu plaque forming units. RNA ribonucleic acid. rpm revolutions per minute. sec second. Taq Thermus aquaticus. u.v. ultraviolet. v/v volume to volume ratio. w/v weight to volume ratio. IIl A adenine. c cytosine. G guanine. T thymine. A Ala Alanine c Cys Cysteine B Asx Asparagine or aspartic acid D Asp Aspartic E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine p Pro Proline Q Gln Glutamine R Arg Arginine s Ser Serine T Thr Threonine V Vat Valine w Trp Tryptophan y Tyr Tyrosine z Glx Glutamine or glutamic acid IV Contents. Abstract. Acknowledgements. Il Abbreviations. Ill Amino Acid and base codes. TV Contents. V List offigures. XII Chapter 1. Introduction. 1 1.1. Summary; arable crops past present and future. 1 1.2. Genus Diabrotica; agricultural pests ofNorth America. 2 1.2.1. D. undecimpunctata life cycle and distribution. 4 1.2.2. Current Diabrotica control technologies. 6 1.3. Insect pesticide and xenobiotic detoxification. 8 1.3.1. Glutathione S-transferases. 10 1.3.2. Cytochrome P450 mixed function oxidases. 12 1.4. Biotechnology and crop protection. 15 1.4.1. Bacillus thuringiensis toxins. 16 1.4.2. Chitinases. 19 1.4.3. Proteinase inhibitors. 22 1.5. Plant proteinase inhibitors. 25 1.5.1. The 20-24 kDa proteinase inhibitors of potato. 26 1.5.2. The Cystatin superfumily. 29 1.6. Insect digestive physiology. 31 1.7. Transport and localisation ofproteinases. 33 1.8. Proteolytic enzymes. 37 1.8.1. Cysteine Proteinases. 38 1.8.2. Aspartic proteinases. 41 1.8.3. Serine proteinases. 43 V 1.8.4. Exopeptidases. 46 1.9. Plant inhibitors of D. undecimpunctata digestive proteinases. 47 1.10. Project aims. 48 Chapter 2. Materials and Methods. 49 2.1. Chemical and solution abbreviations. 49 2.2. Materials and software. 50 2.3. Frequently used media and solutions. 51 2.4. Methods. 55 2.5. Larval D. undecimpunctata culture. 55 2.6. Biochemical methodology. 55 2.6.1. Alimentary tract extraction. 55 2.6.2. Proteolysis assay system. 56 2.6.3. pH dependent proteolysis assays. 57 2.6.4. Proteolysis inhibition assays. 57 2.7. RNA extraction and analysis. 58 2.7.1 Larval gut dissection. 58 2.7.2. Total RNA extraction and purification. 58 2.7.3. mRNA extraction and purification. 58 2.7.4. RNA electrophoresis. 59 2.8. Polymerase chain reaction (PCR). 60 2.8.1. PCR primer design. 60 2.8.2. Reverse-transcriptase PCR. 60 2.8.3. Taq polymerase PCR. 61 2.8.4. Advantage 2 PCR. 62 2.8.5. Cloning PCR products. 62 VI 2.9. DNA manipulation and sequence analysis. 63 2.9.1. DNA electrophoresis. 63 2.9.2. Visualisation and photography ofEtBr stained gels. 63 2.9.3. Silica fines DNA gel slice purification. 63 2.9.4. Restriction endonuclease digestion ofDNA. 64 2.9.5. DNA ligation. 64 2.9.6. DNA sequencing. 65 2.9.7. DNA sequence data analysis. 65 2.10. Bacterial culture and plasmid isolation. 66 2.1 0.1. Overnight cultures. 66 2.1 0.2. Plasmid mini-preparations. 66 2.10.3. Glycerol stocks. 66 2.11. cDNA library construction and screening. 67 2.11 .1. cDNA library generation and evaluation. 67 2.11.2. Digoxigenin labelled cDNA probe generation. 68 2.11.3. cDNA library screening. 69 2.11.4. Antibody detection and visualisation of 71 hybridised probe. 2.11.5. Excision and circularisation ofpTripiEx 2 plasmid. 72 2.12. pET 24a construct preparation, recombinant protein 72 expression and analysis. 2.12.1. pET 24a expression vector construct generation. 72 2.12.2. Recombinant protein expression analysis. 73 2.12.3. Large scale recombinant protein expression. 74 2.12.4. Ni-NT A chromatography. 75 2.12.5. Oryzacystatin purification. 76 2.12.6. Dialysis of protein samples. 77 2.12.7. Protein freeze drying. 77 VII 2.12.8. SDS-PAGE analysis. 77 R.esulllts and! Discussion. Cllnapter3. lBiochemncal cllnarncterisatioi!B of D. undecimpMnctata 79 pD'oteollytic actnvnties. 3.0. Introduction 79 3.1. Results: pH optima of proteolytic activities in D. undecimpunctata 79 alimentary tracts. 3. 2. Results: Inhibition of D. undecimpunctata larval alimentary tract 82 proteolysis by chemical and plant protein proteinase inhibitors. 3. 3. Discussion. 84 Cllnapter4. Molecular isolatioiiD of larval D. undecimpunctata cysteine 86 and aspartic protennases. 4.0. Introduction. 86 4.1. Results: Isolation and identification of aspartic and cysteine proteinase 86 encoding sequence fragments from larval D. undecimpunctata. 4.2. Results: Isolation of cathepsin D and L cON A transcripts from the larval 106 D. undecimpunctata alimentary tract cDNA library. 4.2.1.