Engineering Insect-Resistant Plants by Transgenic Expression of an Insecticidal Spider-Venom Peptide Md. Shohidul Alam Master of Science in Agricultural Chemistry A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience Abstract The insecticidal spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) from the Australian Blue Mountains funnel-web spider Hadronyche versuta is one of the most potent insect-specific neurotoxins isolated to date. Hv1a blocks voltage-gated calcium channels in the insect central nervous system, a mechanism quite distinct from existing chemical insecticides. It induces a slow-onset paralysis that precedes death in a taxonomically wide range of insects. Hv1a's broad spectrum of target insects, novel mode of action, and absence of toxicity to vertebrates makes the Hv1a gene an attractive tool for generating insect- resistant transgenic crops. The oral activity of Hv1a can be enhanced by coupling it to the plant lectin Galanthus nivalis agglutinin (GNA) or with the minor capsid protein of pea enation mosaic virus (CP). Recombinant fusions of Hv1a with GNA were produced using the Pichia pastoris expression system to study the intrinsic insecticidal activity of Hv1a-GNA and GNA-Hv1a fusion proteins. By using injection bioassays with houseflies, we found that the intrinsic insecticidal activity of Hv1a was maintained when it was fused to GNA. Moreover, feeding bioassays with diamondback moth larvae revealed that fusion of Hv1a to GNA, in either orientation, enhances its oral insecticidal activity. In order to generate transgenic plants expressing Hv1a alone or fused to GNA or CP, transformation vectors were constructed by ligating synthesised genes in the pAOV binary vector with the constitutive Cauliflower Mosaic Virus 35S promoter (35S) or the phloem tissue-specific Arabidopsis thaliana SUCROSE TRANSPORTER 2 (SUC2) promoter. Homozygous transgenic Arabidopsis were generated using the floral dip method of Agrobacterium-mediated plant transformation and subsequent herbicide selection. PCR of genomic DNA and western blotting were used to confirm integration of the transgenes and protein expression in the transgenic Arabidopsis respectively. Initial experiments revealed a very high level of mortality of Helicoverpa armigera larvae on wild-type plants due to the presence of endogenous glucosinolates, which masked the insecticidal effects of the transgenes Thus, a new set of transgenic plants was generated using an Arabidopsis cyp79B2 cyp79B myb28 myb29 quadruple mutant that lacks endogenous glucosinolates. Bioassays revealed that H. armigera larvae had a lower level of survival and retarded growth when fed on leaves of transgenic Arabidopsis expressing Hv1a toxins under 35S promoter control compared with those fed on gluc-null control plants. Moreover, larval mortality was higher for plants expressing Hv1a/GNA fusions than those expressing Hv1a or GNA alone. The highest larval mortality, lowest larval weight gain, and lowest level of leaf damage were observed for larvae fed on plants expressing GNA-Hv1a. Mortality was extremely high (~90%) for larvae fed on GNA-Hv1a plants for 15 days. The resistance to cotton bollworms conferred by expression of GNA-Hv1a in transgenic Arabidopsis highlights the potential of Hv1a transgenes as an alternative to harmful chemical insecticides. Moreover, Hv1a transgenes might provide a useful adjunct or alternative to Bt crops, and they might be useful for trait stacking with Bt transgenes. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Publications during candidature Book chapters Herzig, V.; Bende, N. S.; Alam, M. S.; Tedford, H. W.; Kennedy, R. M. & King, G. F. 2014. Chapter Eight - Methods for Deployment of Spider Venom Peptides as Bioinsecticides. In: Tarlochan, S. D. & Sarjeet, S. G. (eds.) Advances in Insect Physiology. Academic Press. Conference abstracts Alam, M.S., Mylne, J. S. and King, G.F. (2013) Spider-venom peptide protects plants from insect pest attack. East Coast Protein Meeting–2013, Coffs Harbour, NSW, Australia. Publications included in this thesis No publications have been incorporated into this thesis. Contributions by others to the thesis Dr Raveendra Anangi and MSc student Mr Mario Donald Bani contributed in part to the design, standardization, and purification of the recombinant fusion proteins described in Chapter 2. Dr Volker Herzig contributed in injection bioassay of houseflies with recombinant fusion proteins described in Chapter 2. Statement of parts of the thesis submitted to qualify for the award of another degree Portions of some of the figures from Chapter 2 were used in an MSc thesis submitted by Mario Donald Bani to The University of Queensland (degree awarded July 2013). Acknowledgements I would like to extend my heartfelt and sincere gratitude to my principal advisor Professor Glenn F. King (Institute for Molecular Bioscience, The University of Queensland, Australia) for his expert supervision, guidance, support and enthusiastic encouragement throughout the course of my PhD. I also wish to express my gratitude, sincere appreciation and indebtedness to my associate advisor Associate Professor Joshua S. Mylne (The University of Western Australia, Australia) for his valuable guidance and cooperation during the research work. I would like to thank Professor Myron Zalucki (School of Biological Sciences, UQ), Dr Steven Reid (SCMB and AIBN, UQ) and Dr Mark Jackson (IMB, UQ) for their guidance and encouragement as members of my candidature committee. I would like to acknowledge those who helped in one way or another during the tenure of my research work and report writing, including Dr Brit Winnen for help with gel electrophoresis and western blotting; Dr Raveendra Anangi for help with recombinant protein expression; Dr Aurelie Chanson for help with recombinant DNA technology and genetic engineering techniques; Dr Lynda Perkins for help with insect bioassays and data analysis; Dr Volker Herzig and Dr Margaret Hardy for help with insect bioassays and data analysis; Dr Elaine Fitches (FERA, York, UK) for providing anti-GNA antibody; and Dr Mark Kinkema (AgBiTech, Queensland, Australia) for providing Helicoverpa armigera eggs. Thanks to all members of the King Group for creating such a warm, friendly and cooperative atmosphere. Particular thanks to Dr Maria Ikonomopoulou, Dr David Morgenstern, Dr Julie Klint, Dr Sandy Pineda Gonzalez, Dr Eivind Undheim, Dr Naushad Shaikh, Mr Niraj Bende, Mrs Darshani Rupasinghe, Mr Carus Lau, Ms Jessie Er and Mr Sebastian Senff for their help, advice, and guidance. I would like to acknowledge all staff of the Queensland Bioscience Precinct and the Institute for Molecular Bioscience (IMB), specially Dr Amanda Carozzi for her help with my scholarship applications and candidature, and Mikiko Miyagi for help in the laboratory. I am also very grateful to the IMB and The University of Queensland (UQ) for funding my RHD candidature via an IMB Postgraduate Award, UQ International Research Tuition Award (UQIRTA), and UQ Research Scholarship (UQRS). Last, but no means least, I would like to express my heartfelt thanks to my family, who have provided limitless love and support over the years. Md. Shohidul Alam Keywords insecticide, insecticidal neurotoxin, insect-resistant transgenic crop, spider-venom peptide, Galanthus nivalis agglutinin, capsid protein, Arabidopsis thaliana, glucosinolates, Helicoverpa armigera, Plutella xylostella Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC Code 100103: Agricultural Molecular Engineering of Nucleic Acids and Proteins, 50% ANZSRC Code 060702: Plant Cell and Molecular Biology, 30% ANZSRC Code 100105: Genetically Modified Field Crops and Pasture, 20% Fields of Research (FoR) Classification FoR Code 1001: Agricultural Biotechnology, 70% FoR Code 0607: Plant Biology, 15% FoR Code 0601: Biochemistry and Cell Biology, 15% Table of Contents Page No. Chapter 1: Introduction………………………………………………………………… 1 1.1 Transgenic crops and food security…………………………………………..……. 1 1.2 Crop losses due