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1. Surender yadav 2009BS41D 2. Introduction to bt toxins ? Bacillus thuringiensis (bt) is a common gram positive, spore-forming, ? ? ? ? soil bacterium. When resources are limited, vegetative bt cells undergo sporulation, synthesizing a protein crystal, the Insecticidal crystal proteins (ICPs) or Cry Proteins. For over 50 years, bt has been applied to crops in spray form as an Insecticide, containing a mixture of spores and the associated protein crystals. The development and commercialization ofInsect-resistant transgenicbtcrops expressing Cry toxins revolutionized the history of agriculture. Benefits ? High specificity and potency, ? Reduction in chemical pesticide applications, ? Increased crop yield. 3. Structure ofbt toxin Cry protein domains •Domain A - Pore formation 6? helices (250 aas) •Domain B -receptor binding domain, ?-sheets (200 aas) •Domain C - ? sandwich (150 aas) protects the toxin from protease 4. bt mode of action 5. bt Toxin Nomenclature: Each bt toxin will be assigned a unique name incorporating four ranks e.g. Cry 1Aa3 •Primary rank - order ofInsect; •Secondary and tertiary ranks - potency and targeting within an order •Quaternary rank- alleles of genes coding for toxins •Classification based on their sequence homology and specificities •CryI genes encoded proteins toxic to lepidopterans; •CryII genes encoded proteins toxic to both lepidopterans and dipterans; •CryIII genes encoded proteins toxic to coleopterans; •CryIV genes encoded proteins toxic to dipterans alone. Crickmore , 1998 6. Introduction to bt plants ? bt plants have genes for the bt toxins engineered to produce ICP toxic to the pest species of concern. ? As the Insect feeds on the bt plant, it ingests the ICP and suffers the same fate as if it ingested leaf tissue sprayed with bt. ? At the end of 2010, an estimated 26.3 million hectares of land were planted with crops containing the bt gene globally(James 2011). The chief advantages to bt plants: ? The pests hiding inside plant parts controlled effectively; ? Multiple sprays are not needed; ? The dose ofbt can be more effectively regulated. ? A disadvantage ofbt plants is that Insect-specific ICPs cannot be changed during a growing season. 7. Insectresistance ? resistance is a genetic change in the Insect pest — that allows it to avoid harm from bt toxins. ? Only two Insect species that have developed resistance to bt foliar sprays under commercial situations — the diamondback moth and the cabbage looper. ? The high and consistent levels of ICP production in the bt plant make them much less favorable for the development ofresistance, ? enough to kill the SS and RS Insect genotypes, and such a dose is impossible to maintain with foliar sprays. ? In the years prior to the development ofresistance- substantial environmental and human health benefits. ? bt cotton has reduced the use of traditional Insecticides. 8. Insectresistance Management ? The practices aimed at reducing the potential for Insect pests to become resistant to a pesticide. ? bt IRM is of great importance because of the threat Insectresistance poses to the future use ofbt plant-incorporated protectants. Risk factors for pest populations evolving btresistance: ? Great genetic diversity in pest populations ? Sexual recombination ? Constitutive production of toxins ? Intense selection pressure on pest population ? IRM is said to be the key to sustainable use of the genetically modified btcrops. 9. Managing btresistance ? The US EPA usually requires a “buffer zone,” or a structured refuge of 20% non-btcrops that is planted in close proximity to the btcrops. ? “High dose plus refugia” ? Plants express enough bt protein to kill all except rare homozygous recessives (RR) ? Heterozygous offspring, produced when homozygous resistant Insects mate with susceptible Insects, are killed ? Refugia “dilute out” heterozygous resistant individuals (RS) ? Assumption: initially, resistant RS mutants are very rare ? As for Insects with recessive alleles for such genes, they are thought to be “diluted out” by susceptible Insects from the refugia. 10. Why does adding susceptible plants (refuges) slow evolution ofbtresistance? ? btresistance as recessive: need to lose/mutate both copies of the receptor gene to become resistant. ? Refuge is more effective the less dominant that btresistance is because the RS genotypes don’t survive well. ? The development ofresistance is driven by the initially very rare RR genotypes, but for a long time they only have the RS types to mate with. ? Planting refuges minimize the differential in fitness between the more and less resistant genotypes will slow evolution ofresistance. 11. Seed mixtures strategies ? Seed mixture strategy involves random mixing of 20% non-bt plants among bt plants. ? Poor compliance of the Indian farmers to grow refuge crop in bt cotton fields. ? Commercial packets ofbt seeds will also contain non-transgenic seeds premixed. ? However, the strategy of seed mixtures can become ineffective and unproductive if the frequency of resistant Insect pests has already become unmanageable. 12. Methods of growing bt and non-bt plants for btresistance management. Structured refugia contain two rows of non-bt plants (bold dotted lines) for every eight rows ofbt plants Seed mixture strategy involves random mixing of 20% non-bt plants (bold dots) among bt plants. Vageeshbabu ,2011 13. Evolution ofresistance to bt toxin ? Although there were no cases ofInsects developing resistance to bttransgenic plants in the field, ? laboratory populations of Cry1A-resistant DBM have been shown to be able to survive on high levels of Cry1Ac ? In cases where resistance to btcrops has evolved quickly, one or more conditions of the refuge strategy have not been met. 14. First documented case of pest resistance to bt cotton ? Tabashnik, (2008) observed that the frequency of resistant alleles has increased substantially and that there is field-evolved bt toxin resistance in bollworm, Helicoverpa zea (Boddie), in the United States. ? The concentration of Cry1Ac in bt cotton was not high enough to kill the hybrid offspring produced by matings between susceptible and resistant H. zea. ? Thus, the so-called “high dose” requirement was not met . ? In a related case, failure to provide adequate refuges of non-bt cotton allowed the pink bollworm to evolve resistance to bt cotton in India (Bagla 2010). 15. Field-Evolved resistance to bt Maize by Western Corn Rootworm (Gassmann,2011) ? Fields experiencing severe rootworm feeding contained Cry3Bb1 maize. ? These displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays. ? A significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. ? However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these bt toxins. ? Insufficient planting of refuges and non-recessive inheritance ofresistance may have contributed to resistance. 16. ? To engineer crops that express at least two toxic compounds that act ? ? ? ? ? independently, so that resistance to one does not confer resistance to the other. This approach, called gene pyramiding, became a commercial reality in 2003 with the introduction of Bollgard II, A transgenic cotton plant that expresses the original bt protein, Cry1Ac, and a second bt protein, Cry2Ab. The two proteins act independently in that they bind to different receptors in the Insect’s midgut. Insects homozygous for one resistance gene are rare, Insects homozygous for multiple resistance genes are extremely rare (Karim et al. 2000) A species cannot easily evolve resistance to both toxins because that would require two simultaneous, independent mutations in genes encoding the receptors (Jackson et al., 2003). 17. SmartStax corn ? The multitoxin btcrops are designed to help delay resistance and to kill a broader spectrum ofInsect pests. ? SmartStax corn has eight GE traits ‘stacked’ together – 6 for Insectresistance (bt) and 2 for herbicide tolerance. ? Tolerance to aerial pests (three bt genes): Cry 1A.105 (Monsanto), Cry 2Ab2 (Monsanto) and Cry 1F (Dow). ? Tolerance to subsoil pests (three bt genes): Cry 3Bb1 (Monsanto), Cry 34Ab1 (Dow) and Cry 35Ab1 (Dow). ? Tolerance to herbicides (two genes): Glyphosate (Monsanto) and Glufosinate (Dow). 18. Limitations of Gene Pyramid ? Greater the number of genes, more plant protein will be diverted away from creating useful yield. ? This scenario sets the risk of significant agronomic and yield penalties which may make the variety unattractive to the grower. ? One toxin can bind to several sites. Such a scenario can lead to the development of cross resistance or multiple resistanceof an Insect in cases where it was never exposed to the original toxin. 19. Mechanisms ofbt toxin resistance ? bt works by binding to toxin receptor (cadherin), which triggers cleavage ofbt protein ? bt-resistant Insects express mutated cadherin proteins that do not bind toxins. ? Modified toxins can make resistant cadherinmutated Insects susceptible again (Soberon et al, Science, 2007) ? Toxins with independent actions bind to different sites ? Multiple resistance: one toxin can bind to several sites (e.g., Insect develops resistance to multiple bt toxins after repeated exposure to one) 20. ? Cadherin gene silencing with RNAi in tobacco hornworm resulted in ? ? ? ? ? ? reduced susceptibility to the bt toxin Cry1Ab, confirming cadherin’s role in bt toxicity. The binding of protease-activated toxin to cadherin is essential for the removal of helix ?-1, which in turn promotes oligomerization. Modified Cry1Ab and Cry1Ac toxins lacking helix ?-1 (referred to as Cry1AbMod and Cry1AcMod) could form oligomers without cadherin. The modified toxins killed cadherin-silenced hornworm and bt-resistant pink bollworm that had cadherin deletion mutations. Conversely, against susceptible larvae, the native toxins were more potent than the modified toxins. This implies that modified toxins had lower stability in the midgut, reduced oligomer-forming ability, or reduced ability of oligomers to ultimately cause mortality. These findings demonstrate that the modified bt toxins may be useful against pests resistant to standard bt toxins. 21. Effect of RNAi on cadherin protein expression 22. Responses of susceptible (APHIS-S) and resistant (AZP-R) pink bollworm larvae 23. Efficacy of genetically modified bt toxins against Insects with different genetic mechanisms ofresistance. ? Relative to native toxins, the potency of modified toxins was >350-fold higher against resistant strains of DBM(Px) and European corn borer (On) in which resistance was not linked with cadherin mutations. ? Conversely, the modified toxins provided little or no advantage against some resistant strains of three other pests with altered cadherin. ? Independent of the presence of cadherin mutations, the relative potency of the modified toxins was generally higher against the most resistant strains. Tabashnik (2011) 24. resistance to six species ofInsect pests 25. Potency of modified bt toxins relative to native bt toxins ? The reductions in resistance ratio for modified toxins relative to native ? ? ? ? toxins occurred because modified toxins were more potent than native toxins against resistant strains in four of six cases and less potent than native toxins against susceptible strains in all cases For example, against the resistant strain of DBM, potency was >350fold higher for Cry1AbMod than for Cry1Ab, and >540-fold higher for Cry1AcMod than for Cry1Ac. However, against the susceptible strain of DBM, each modified toxin was less potent than the corresponding native toxin. Cry1AcMod was less potent than Cry1Ac against resistant strains of Bollworm and Sugar cane borer. 26. Suppressing resistance to bt cotton with sterile Insect releases ? An alternative strategy for delaying pest resistance to btcrops where sterile Insects are released to mate with resistant Insects and refuges are scarce or absent. ? Unlike the refuge strategy, this approach does not require maintenance of pest populations and thus compatible with eradication efforts. ? During a large scale, four-year field deployment of this strategy in Arizona, resistanceof pink bollworm to bt cotton did not increase. ? A multitactic eradication program that included the release of sterile moths reduced pink bollworm abundance by >99%, while eliminating Insecticide sprays against this key invasive pest. Tabashnik (2010) 27. ? Many plant secondary metabolites are toxic to or repel Insects, enabling host plants to escape from Insect herbivores (Gatehouse 2002). ? To counteract plant defenses, Insects have developed adaptive mechanisms, which often involve a set of genes whose products metabolize the chemicals from plants (Wittstock et al. 2004). ? Most cotton cultivars accumulate gossypol in both aerial tissues and roots, and these phytoalexins form a chemical arsenal against herbivorous. 28. ? They isolated a P450 monooxygenase gene, CYP6AE14, from Helicoverpa armigera ? Expression of CYP6AE14 was induced by gossypol, and its expression level was correlated with larval growth when gossypol was present in the diet. ? When bollworms were fed on transgenic Arabidopsis plants producing dsRNA against CYP6AE14 (dsCYP6AE14), expression of CYP6AE14 was suppressed; ? After transferring to a gossypol-containing diet, the larvae showed decreased tolerance to gossypol . ? Therefore, cotton plants are engineered to express dsCYP6AE14, which indeed acquired enhanced resistance to cotton bollworms. 29. Effect of T1 transgenic cotton on larvae growth 30. qRT-PCR analysis of CYP6AE14 transcripts in midgut of second-instar larvae 31. Effect of ds6-3 T2 plants on larvae growth 32. transgenic cotton plants were less damaged by bollworms than the control 33. ? The dsCYP6AE14 cotton plant did have deleterious effects on bollworms, but was not lethal. ? If multiple genes involved in the P450 complex were targeted by RNAi, the deleterious effects would be magnified. 34. VipCot cotton ? EPA has conditionally registered a new cotton plant-incorporated protectant, VipCot, of Syngenta Seeds Inc. ? VipCot produces the modified Cry1Ab and Vip3Aa19 proteins derived from Bacillus thuringiensis (bt) to control lepidopteran pests. ? The Vip3Aa19 protein expressed in VipCot cotton provides a unique mode of action. ? When coupled with modified Cry1Ab in VipCot, the proteins have the potential to provide benefits for IRM including: ? High-dose (for both proteins expressed together) against the major target pests, ? Lack of cross-resistance (Vip3Aa19), ? The potential to delay development ofresistance in other cotton varieties expressing Cry toxins. ? VipCot (COT102 x COT67B) was developed by conventional breeding of COT102 (Vip3Aa19) plants with COT67B (modified Cry1Ab) plants. 35. ? The Bacillus thuringiensis vegetative Insecticidal protein Vip3A lyses midgut epithelium cells of susceptible Insects. ? Vip3Aa19 protein is intended to control several lepidopteran pests of cotton including tobacco budworm, cotton bollworm, fall armyworm, beet armyworm and cabbage looper. ? There is no evidence of either a synergistic or antagonistic interaction between Vip3Aa19 and modified Cry1Ab in cotton bollworm or tobacco budworm. ? It demonstrate that data on the individual events and individual proteins can be used to support the safety of the COT102 x COT67B (VipCot) combined product. 36. futureofbtcrops in INDIA ? Whether GM Food is required or not for the country? ? if the perception is not clear; it is going to affect ongoing research. ? Need for additional biosafety studies to assess the safety ofbt protein? ? Need for setting up an independent GMO testing facility devoid of conflict of interest? ? Limited release ofbt seeds to identified farmers under strict expert supervision should be undertaken to evaluate its performance in public space? ? The Biotechnology Regulatory Authority of India Bill is approved by the Government which will replace GEAC. ? The Bill seeks to create a new body to regulate research, manufacture, import and use of products of modern biotechnology. 37. ? The adoption of biotech crops in the next five years period will be dependent mainly on three factors: ? the timely implementation of appropriate, responsible and cost/time- effective regulatory systems; ? strong political will and support; ? a continuing wave of improved biotech crops that will meet the priorities of industrial and developing countries in Asia, Latin America and Africa. 38. Conclusion ? Together with the reduction of pesticide application and cost reduction, btcrops have brought tremendous benefit to both the environment and farmers . ? Expanded use oftransgeniccrops for Insect control will likely include more varieties with combinations of two or more bt toxins, novel bt toxins such as VIP ? Modified bt toxins that have been genetically engineered to kill Insects resistant to standard bt toxins. ? transgenic plants that control Insects via RNA interference are also under development. 39. ? Increasing use oftransgeniccrops in developing nations is likely, with a broadening range of genetically modified crops and target Insect pests . ? Incorporating enhanced understanding of observed patterns of field- evolved resistance into futureresistance management strategies can help to minimize the drawbacks and maximize the benefits of current and future generations oftransgeniccrops.