VIEWS: 145 PAGES: 47 POSTED ON: 4/4/2011
The genetic manipulation of PEST resistance Lum Mok Sam RT 2014 Agriculture Biotechnology Introduction • 13% of the potential world crop yield is lost to pest • Plant pests range from nematodes birds mammals • Insect pests cause a MAJOR proportion of the total pest damage to crops RT 2014 Agriculture Biotechnology The nature & scale of insect pest damage to crops • Adult insects (feed off plants) • Insect larvae RT 2014 Agriculture Biotechnology Major classes of insect that cause crop damage Orders: • Lepidoptera – butterflies & moths • Diptera – flies & mosquitoes • Orthoptera – grasshopper, crickets • Homoptera – aphids • Coleoptera – beetles RT 2014 Agriculture Biotechnology Common insect pests of major crops Insert species Common name of pest Order Crops affected Ostrinia nubilalis European corn borer Lepidoptera Maize Heliothis virescens Tobacco budworm Lepidoptera Tobacco, cotton Hekiothis armigera Old world bollworm Lepidoptera Cotton, tomato Tomato fruit worm Helicoverpa zea Cotton bollworm Lepidoptera Cotton Manduca sexta Tobacco hornworm Lepidoptera Tobacco, tomato, potato Spodoptera littoralis Cotton leaf worm Lepidoptera Maize, rice, cotton, tobacco Leptinotarsa decemlinata Colorado beetle Coleoptera Potato Callosobruchus maculatus Cowpea seed beetle Coleoptera Cowpea, soybean Tribolium confusum Confused flour beetle Coleoptera Cereal flours Locusta migratoria Locust Orthoptera Grasses Nilaparvata lugens Brown plant hopper Homoptera Rice RT 2014 Agriculture Biotechnology GM strategies for insect resistance 1.The use of bacterial insecticidal genes to provide protection from pest damage • The Bacillus thuringiensis approach 2.The use of endogenous plant protection mechanism • A Copy Nature approach RT 2014 Agriculture Biotechnology Bacillus thuringiensis approach RT 2014 Agriculture Biotechnology • B. thuringiensis • Discovered by Ishiwaki in 1901 in diseased silkworms • Produces an insecticidal crystal protein (ICP) RT 2014 Agriculture Biotechnology Insecticidal crystal protein (ICP) • Form inclusion bodies of regular bipyramidal/ cuboidal crystals during sporulation • One of several classes of endotoxins produced by the sporulating bacteria • Originally classified as δ-endotoxins RT 2014 Agriculture Biotechnology cry genes • Carried on plasmid • Belong to a superfamily of related genes RT 2014 Agriculture Biotechnology Classification of ICP genes of B. thuringiensis cry gene families Protein size B. Thuringiensis subspecies/ Susceptible insect class (kDa) strain of holotype cry1Aa(1-14) 133 kurstaki Lepidoptera cry1Ab(1-16) 130 berliner Lepidoptera cry1Ac(1-15) 133 kurstaki Lepidoptera cry1Ad-g 133 Aizawai Lepidoptera cry1Ba(1-4) 140 kurstaki Lepidoptera cry1Ba-g 1340 EG5847 Lepidoptera cry1Ca(1-8) 134 entomocidus Lepidoptera cry1Cb(1-2) 133 galleriae Lepidoptera cry1Da(1-2) 132 Aizawai Lepidoptera cry1Db(1-2) 131 BTS00349A cry1Ea(1-6) 133 Kenyae Lepidoptera cry1Eb1 134 aizawai Lepidoptera cry1Fa(1-2) 134 aizawai Lepidoptera cry1Fb(1-5) 132 morrisoni cry1Ga(1-2) 132 BTS00349A cry1Gb(1-2) 133 wuhanensis Lepidoptera cry1Ha-b 133 BTS02069AA cry1Ia(1-9) 81 kurstaki Lepidoptera cry1Ib-e 81 entomocidus Lepidoptera & Coleoptera cry1Ja-d 133 EG5847 Lepidoptera RT 2014 Agriculture Biotechnology cry1Ka1 137 morrisoni Lepidoptera cry2Aa(1-10) 71 kurstaki Lepidoptera & Diptera cry2Ab(1-5) 71 kurstaki Lepidoptera cry2Ac(1-2) 70 shanghai Lepidoptera cry3Aa(1-7) 73 tenebrionis Coleoptera cry3Ba(1-2) 75 tolworthi Coleoptera cry3Bb(1-3) 74 EG4961 Coleoptera cry3Ca1 73 kurstaki Coleoptera cry4Aa(1-3) 135 israelensis Diptera cry4Ba(1-5) 128 israelensis Diptera cry5Aa1 152 darmstadiensis Nematodes cry5Ab1 142 darmstadiensis Nematodes cry5Ac1 135 PS86Q3 Hymenoptera cry5Ba1 140 PS86Q3 Hymenoptera cry6Aa(1-2) PS52A1 Nematodes cry6Ba1 PS69D1 Nematodes cry7Aa1 129 galleriae Coleoptera cry7Ab(1-2) 130 dakota Coleoptera cry8A-D 131 kumamotoensis Coleoptera cry9Aa(1-2) 130 galleriae Lepidoptera cry9Ba1 galleriae Lepidoptera cry9Ca1 130 tolworthi Lepidoptera cry9Da(1-2) 132 japonensis cry10Aa1 78 israelensis Diptera cry11Aa(1-2) 72 Israelensis Diptera cry11Ba-b 81 Jegathesan Diptera cry12-40 various Various various RT 2014 Agriculture Biotechnology The range of ICPs in individual B. thuringiensis strains B.t. subspecies Crystal protein & strains aizawai Cry1Aa, Cry1Ab, Cry1Ad, Cry1Ca, Cry1Da, Cry1Eb, Cry1Fa, Cry9Ea, Cry39Aa, Cry40Aa entomocidus Cry1Aa, Cry1Ba, Cry1Ca, Cry1Ib galleriae Cry1Ab, Cry1Ac, Cry1Da, Cry1Cb, Cry7Aa, Cry8Da, Cry9Aa, Cry9Ba israelensis Cry10Aa, Cry11Aa japonensis Cry8Ca, Cry9Da jegathesan Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa kenyae Cry2Aa, Cry1Ea, Cry1Ac kumamotoensis Cry7Ab, Cry8Aa, Cry8Ba Kurstaki HD-1 Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ia, Cry2Aa, Cry2Ab Kurstaki HD-73 Cry1Ac Kurstaki NRD-12 Cry1Aa, Cry1Ab, Cry1Ac morrisoni Cry1Bc, Cry1Fb, Cry1Hb, Cry1Ka, Cry3Aa tenebrionis Cry3Aa tolworthi Cry3Ba, Cry9Ca wuhanensis Cry1Bd, Cry1Ga, Cry1Gb RT 2014 Agriculture Biotechnology Cry proteins • Tend to cluster as either • Large (~130 kDa) protein or • Small (~70 kDa) protein • Difference in size between different subfamilies • Share a common action core comprising 3 domains (I, II & III) RT 2014 Agriculture Biotechnology Comparison of the structures of different classes of Cry protein Activated toxin Cry1A N I II III C Cry1B I II III Cry3A I II III Truncated forms in transgenic plants 0 600 1200 Amino acid residues RT 2014 Agriculture Biotechnology • The N-terminal end of each gene has a similar organization • Great difference in overall length • The N- & C-terminal extensions are trimmed by insect gut proteases to release the active toxin • In some plants, a truncated, active form of the protein is produced directly RT 2014 Agriculture Biotechnology Ribbon model of Cry1Aa toxin molecule Domain III Domain I Domain II RT 2014 Agriculture Biotechnology • Domain I • At the N-terminal end • Comprises a series of α-helices arranged in a cylindrical formation • involved in membrane insertion & pore formation • Domain II • Comprises a triple β-sheet • Involved in receptor reorganization • Domain III • β-sandwich • Involved in • Protection from degradation • Toxin/bilayer interactions • Receptor binding RT 2014 Agriculture Biotechnology δ-endotoxins • Involves a specific interaction between the protein & the insect larva midgut • Extremely toxic • Lethal to susceptible insect larvae at relatively low [ ] • Toxicity to mammals is extremely low RT 2014 Agriculture Biotechnology Action mode of δ-endotoxins δ-endotoxins Ingest by an insect larva Protein crystals are solubilised in insect larva midgut Larger protein (e.g. 130 kDa Cry1 group) are proteolytically cleaved Active 55-70 kDa active fragment of the protein released Interacts with high affinity receptors in the midgut brush-border membrane Open cation-selective pores in the membrane Flow of cations into the cells Osmotic lysis of the midgut epithelium cells Destruction of the midgut epithelium cells RT 2014 Agriculture Biotechnology The conditions in the insect midgut • Vary according to insect class • Midgut of • Lepidoptera & Diptera: mildly alkaline • Coleoptera: more alkaline/ acidic • Different conditions favour the solubilisation & activation of different Cry subfamilies • Individual Cry proteins are active against particular insect larvae RT 2014 Agriculture Biotechnology The use of ‘Bt’ as a biopecticide • B. thuringiensis spores biopecticide • Isolated crystals Useful for the rapid development of the ‘Bt’ strategy to genetic manipulation Shorthand for a crop transformed with • cry gene – Bt cotton • Cry proteins – Bt protein RT 2014 Agriculture Biotechnology Bt-based genetic modification of plants Modified cry gene enhance expression Protection against damage by susceptible insect larvae RT 2014 Agriculture Biotechnology Commercialization of Bt technology Company Trade Bt Crops Insect pests name protein Monsanto New-Leaf Cry3A Potato Colorado beetle discontinued Monsanto Bollgard Cry1Ac Cotton Tobacco budworm, cotton bollworm, pink bollworm Currently Monsanto YieldGard Cry1Ab Maize European corn borer commercially DeKalb Bt-Xtra Cry1Ac Maize European corn borer grown in the Aventis StarLink Cry9C Maize European corn borer USA Mycogen Herculex 1 Cry1F Maize European corn borer Monsanto pending Cry3Bb Maize Corn rootworm larvae The specificity of Cry proteins permits the targeting of specific pests by particular transgene, and that different crops may have different cry gene inserted RT 2014 Agriculture Biotechnology The problem of insect resistance to Bt Repeated growing of Bt crops in the same area Provide the selective advantage to accelerate the appearance of a resistant pest population RT 2014 Agriculture Biotechnology • Mechanism • the specific binding involved in the mechanism of action of the Cry proteins • only require a small number of significant mutations in the insect gene coding for the receptor protein to greatly reduce the binding of a particular Cry protein • Appear within a few generations RT 2014 Agriculture Biotechnology Strategies for countering the build-up of insect resistance 1. To use more than one transgene – pyramiding • Transgenes are successfully ‘stacked’ by conventional crosses between different transgenic lines • To avoid cross-resistance to two different Bt genes pyramid Bt genes with unrelated resistance genes RT 2014 Agriculture Biotechnology 2. To further enhance the effectiveness & range of activities of cry genes • e.g. domain engineering to produce chimeric proteins Bt & other GM approaches to insect resistance cannot be viewed as a ‘magic bullet’ to permanently eliminate the threat of insect damage RT 2014 Agriculture Biotechnology Integrated pest management – IPM • Crop rotation Rotating Bt crops with non-Bt crops May prevent the build-up of resistance in the insect RT 2014 Agriculture Biotechnology High dose/ refuge-resistance management scheme Non-transgenic Non-transgenic crop Bt crop crop rr rr rr rr RR rr rr Bt crops expressing a high dose of Bt protein Rr RT 2014 Agriculture Biotechnology The environmental impact of Bt protein • Pollen from Bt maize might be toxic to the larvae of the Monarch butterfly e.g. Bt176 One of North America’s most colorful & familiar natives RT 2014 Agriculture Biotechnology The ‘Copy Nature’ strategy RT 2014 Agriculture Biotechnology • Involves a rational approach to the development of pest-resistant crops 1.Identification of leads • Identification of resistant plants in nature • Discovery of plant genes that could confer resistance to insect damage 2.Protein purification • Purification of proteins with insecticidal properties RT 2014 Agriculture Biotechnology 3.Artificial-diet bioassay • To determine the activity of the isolated protein against the target insect pest by performing feeding assays in the laboratory 4.Mammalian toxicity testing • Prior to any insertion of the gene into a crop plant, the toxicity of the protein against mammals should be tested RT 2014 Agriculture Biotechnology 5.Genetic engineering • Transfer of isolated gene into crop plants • Pest-resistant construct: choice of promoter 6.Selection & testing Transformation Selection of transgenic plants Confirmation of transformation Inheritance of transgene in generations Testing of expression levels • Effectiveness of the construct: evaluated by insect feeding assay RT 2014 Agriculture Biotechnology Promoters used with insect resistance genes Promoter Origin Expression Insecticidal Plant site protein Mannopine synthase Agrobacterium Most plant Cry1Ab Tobacco, TR Ti plasmid tissues potato Phytohaemagglutinin Bean Seed A-AI-Pv Pea, adzuki (PHA-L) bean, tobacco CaMV 35S Cauliflower Most plant Most proteins Most plants mosaic virus tissues Sucrose synthase Rice Phloem GNA Tobacco (RSs1) Metallothionein-like Maize Root Cry1Ab Maize (MT-L) preferred Phosphoenolpyruvate Maize Green Cry1Ab Maize, rice carboxylase (PEPC) tissue RT 2014 Agriculture Biotechnology Promoter Origin Expression Insecticidal Plant site protein Pollen-specific Maize Pollen Cry1Ab Maize Tryptophan synthase Maize Pith preferred Cry1Ab Maize α-subunit (trpA) Ubiquitin-1 (Ubi-1) Maize All plant Cry1Ac Rice organs Proteinase inhibitor II Maize Wound Pot PI-II, ipt Rice, (Pot PI-II) inducible tobacco, tomato rRNA operon (Prrn) Potato Chloroplasts Cry1Ac Tobacco Actin-1 (Act-1) Rice All plant CpTI Rice organs Pathogenesis-related Tobacco Chemically Cry1Ab Tobacco protein-1a (PR-1a) induced RT 2014 Agriculture Biotechnology 7.Biosafety • Effect of the transgene on • Crop yield Evaluated in • Insect damage field trials • Wider ecosystem • Aim: insect pest control which is relatively sustainable and environmentally friendly RT 2014 Agriculture Biotechnology • Recognizes a complex interplay in biological communities between plants, animals, microbes, the soil and the physical environment • Host-plant resistance to pest is universal: 2 major categories 1. Horizontal resistance 2. Vertical resistance RT 2014 Agriculture Biotechnology 1. Horizontal resistance • Usually polygenic • Not involve gene-gene matching • Usually durable RT 2014 Agriculture Biotechnology 2. Vertical resistance • Typically arises from one major gene with a high level of expression • Involve gene-gene matching • Normally occurs in plants to provide • A buffer during short-term changes in the level • Type of insect damage • Unlikely to be durable RT 2014 Agriculture Biotechnology Plant insecticidal genes used to engineer pest resistance Plant gene Encoded Plant of Target Transformed plants protein origin insects Protease Inhibited inhibitors protease C-II Serine Soybean Coleoptera, Oilseed rape, poplat, potato, protease Lepidoptera tobacco CMe Trypsin Barley Lepidoptera Tobacco CMTI Trypsin Squash Lepidoptera Tobacco CpTI Trypsin Cowpea Coleoptera, Apple, lettuce, oilseed rape, Lepidoptera potato, rice strawberry, sunflower, sweet potato, tobacco, tomato, wheat 14K-CI Bifunctional Cereals Tobacco serine protease and α- amylase RT 2014 Agriculture Biotechnology Plant gene Encoded Plant of Target Transformed plants protein origin insects MTI-2 Serine Mustard Lepidoptera Arabidopsis, tobacco protease OC-1 Cysteine Rice Coleoptera, Oilseed rape, poplat, tobacco protease Homoptera PI-IV Serine Soybean Lepidoptera Potato, tobacco protease Pot PI-I Proteinase Potato Lepidoptera, Petunia, tobacco Orthoptera Pot PI-II Proteinase Potato Lepidoptera, Birch, lettuce, rice, tobacco Orthoptera KTi3, SKTI Kunitz Soybean Lepidoptera Potato, tobacco, rice trypsin PI-I Proteinase Tomato Lepidoptera Alfalfa, tobacco, tomato PI-II Proteinase Tomato Lepidoptera Tobacco, tomato RT 2014 Agriculture Biotechnology Plant gene Encoded Plant of Target insects Transformed plants protein origin α-Amylase inhibitors 1-AI-Pv α-amylase Common Coleoptera Azuki bean, pea, tobacco bean WMAI-I α-amylase Cereals Lepidoptera Tobacco 14K-Cl Bifunctional Cereals Tobacco serine protease and α-amylase RT 2014 Agriculture Biotechnology Plant gene Encoded Plant of Target Transformed plants protein origin insects Lectins GNA Lectin Snowdrop Homoptera, Grapevine, oilseed rape, potato, Lepidoptera rice, sweet potato, sugarcane, sunflower, tobacco, tobacco P-lec Lectin Pea Homoptera, Potato, tobacco Lepidoptera WGA Agglutinin Wheat Lepidoptera, Maize germ Coleoptera Jacalin Lectin Jack fruit Lepidoptera, Maize Coleoptera Rice lectin Lectin Rice Lepidoptera, Maize Coleoptera RT 2014 Agriculture Biotechnology Plant gene Encoded Plant of Target Transformed plants protein origin insects Others BCH Chitinase Bean Homoptera, Potato Lepidoptera Peroxydase Anionic Tobacco Lepidoptera, Sweet gum, tobacco, tomato peroxydase Coleoptera, Homoptera Chitinase Chitinase Oilseed rape TDC Tryptophan Catharanthus Lepidoptera Tobacco decarboxylase roseus RT 2014 Agriculture Biotechnology Insect resistant crops and food safety • Certain protease inhibitors and lectins are known to have toxic effects in mammals e.g. the snowdrop lectin GNA – Potatoes carrying this transgene might be responsible for changes to the gut lining of rats (The Pusztai affair) RT 2014 Agriculture Biotechnology
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