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Inactivation of Allergens and Toxins

Inactivation of Allergens and Toxins. Transgenic Plants for Food Security in the Context of Development Rome, 15-19 May 2009. Piero Morandini Dept. of Biology Milan University (Italy). The dangers of nature and food Gene inactivation strategies Manipulating crops (toxins)

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Inactivation of Allergens and Toxins

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  1. Inactivation of Allergens and Toxins Transgenic Plants for Food Security in the Context of Development Rome, 15-19 May 2009 Piero Morandini Dept. of Biology Milan University (Italy)

  2. The dangers of nature and food Gene inactivation strategies Manipulating crops (toxins) Manipulating crops (allergens) Transgenic vs. classical approaches Consequences & conclusions Most of the material presented is the work published by other groups Outline

  3. Nature: a ‘mine field’ Toxic substances abound in nature, both in cultivated and wild plants With time we learned how to avoid some (or at least limit their intake), inactivate others through various processes: Proper storage Cooking (e.g. heat inactivation) Food processing (e.g.: maceration, fermentation) precise mapmetal detector Knowledge handed through culture and technology (to detect, avoid & inactivate) are crucial for survival Technology and knowledge buffer us from the toxic effect of nature

  4. A field trip into the ‘mine field’

  5. LD50=30 mg/kg Solasonine Solasonine accumulates in Solanum sodomeum http://kanaya.naist.jp/knapsack_jsp/image.jsp?word=C00002265

  6. March 27, 1925 340-341 http://depthofprocessing.blogspot.com/2009 /05/are-potato-peels-nutritious.html Poisoning and Toxicology Handbook by Leikin & Paloucek 4th edition, Informa Health Care, 2007 ISBN 1420044796, 9781420044799 http://commons.wikimedia.org/wiki/File:Potato_sprouts.jpg Solanine abounds in green parts, sprouts and diseased potatos

  7. A matter of dose (threshold)  reduction below a threshold considered safe

  8. http://www.springerlink.com/content/37827612x62xp348/ The issue of toxic substances in plants is not new nor is gone Cultivated plants have less toxins than wild relatives (e.g. potato) What happened? Mutants selected by human & animal tests Trial and error (or trial and death) Many crops still produce low levels of toxins (capacity is there!) Their content may increase by breeding or spontaneously: e.g. Potato, Celery, Zucchini (courgette)…

  9. Bottle gourd Celery How to reduce toxic substances in plant?  Target the gene(s) Gene  RNA  Protein Toxins are either proteins or are produced through proteins http://nonsense123.files.wordpress.com/2008/06/bottle-gourd.jpg Which approaches? Zucchini Blackjack http://www.people.cornell.edu/pages/kjc34/distribution.html http://www.growfruitandveg.co.uk/grapevine/vegging-out/courgettes-doing-my-head_18095.html

  10. Degradation product Gene inactivation strategies a) inactivate a protein (enzyme) Regulator b) increase toxin degradation c) target a regulator of the synthesis Halkier (2006) Annu. Rev. Plant Biol. 57:303-33

  11. Tools of the tradeto inactivate (plant) genes • ‘Classical’ mutation (base change, insertion, deletion…) • Insertional mutagenesis (transposons or T-DNA) • RNA mediated (antisense, RNAi, miRNA, hpRNA…) collectively known as post-transcriptional gene silencing (PTGS), often involving epigenetic changes Different methods may end up exactly in the same result (inactivation of a gene) and the same change at the DNA level

  12. Each method has pros & cons • None suites all situations • Hard or impossible distiguish natural / non-natural

  13. Transcription Direct gene inactivation • The gene is mutated (becomes non functional) • RNA missing or aberrant • Protein missing or non functional • Usually irreversible Protein toxin ‘good’ phenotype

  14. ‘Classical’ mutation • Mutations arise spontaneously in any organism (endogenous or environment) • Frequency can be enhanced by various treatments: UV, X and γ-rays, chemical mutagens, and mitogens (indirectly) • Crop plants accumulated many mutations Just one base change out of 15,000 Konishi et al., (2006) Science 312:1392-1396

  15. T-DNA STOP Gene = meaningful sentence ATG Insertional mutagenesis Transposons: genetic elements able to jump around in the genome Retroviruses: virus making new copies (through RNA) able to integrate into the genome. T-DNA: bacterial DNA inserted into the plant genome Inactivate genes by: A large bit of DNA ‘breaks’ the gene  meaning is lost or altered

  16. Antisense RNA Transcription Duplex formation Protein toxin Transcription Block of translation Protein Indirect gene inactivation • The gene is intact but its expression inhibited • RNA is either missing, destroyed or non functional • Another gene is responsible for the change • Only knowledge of the sequence required toxin ‘good’ phenotype

  17. Gene silencing petunia expressing a maize gene The presence of multiple copies of the maize gene causes partial or complete silencing of an endogenous gene

  18. Gene to be silenced RNA-mediated • Once you know the sequence of a gene, it becomes easy to inactivate • only that gene • only in certain tissue/organ Mansoor et al., (2006) Trends Plant Sci. 11:559-65.

  19. Gossypol, a problem • Cotton produces 1.65 kg of seed for 1 kg of fiber • Due to gossypol, a cardio- and hepatotoxic terpenoid  seed unfit for consumption by humans and monogastric animals) • Seed contains 21% oil and 23% high-quality protein Sunilkumar et al. (2006) P.N.A.S. 103:18054–18059

  20. Cottonseed may help feed the world • Used as feed for ruminant animals (whole seeds or meal after oil extraction) • 44 million metric tons (Mt) of cottonseed (9.4 Mt of protein) • Could fulfill protein requirements of half a billion people each year (50 g/day rate) Sunilkumar et al. (2006) PNAS 103:18054-9

  21. RNAi inactivating δ-cadinene synthase in the seed Chlorophyll Carotenoids … Proposed biosynthetic pathway of gossypol Sunilkumar et al. (2006) PNAS 103:18054-9

  22.  a strong reduction of gossypol in seed Levels of gossypol (mg/mg seed) for each individual seed Sunilkumar et al. (2006) PNAS 103:18054-9

  23. Transgenic seed exhibits a large reduction in Gossypol level. A monogenic trait: the reduced gossypol trait cosegregates with the transgene  Much more predictable, stable, specific Sunilkumar et al. (2006) PNAS 103:18054–18059

  24. Transgenic vs. conventional • A glandless mutant was obtained with conventional strategies. Varieties with this trait were a failure under field conditions (extraordinarily susceptible to a host of insect pests) • Terpenoids protect the plant from both insects and pathogens • The transgenic approach achieved a goal classical breeding was unable to obtain (specific reduction in seed) Targeted gene silencing can be used to modulate biosynthetic pathways in a specific tissue to obtain a desired phenotype. Impossible by traditional breeding  Texas A&M University and U.S. Department of Agriculture Sunilkumar et al. (2006) PNAS 103:18054–18059

  25. Take home message (I) “[this] approach…not only improves food safety but also provides an additional and potentially extraordinary mean to meet the nutritional requirements of the growing world population without having to increase either crop yields or acreage planted” (Sunilkumar et al., 2006) “Our hope is to get through regulatory approval process in the U.S. first. However, it takes $50-100 million to go through the process. At this point, we don't know where the money is going to come from, but we are exploring various possibilities. Getting U.S. approval will make it easier to then get permit in other countries. We will be especially interested in some African countries and some Asian countries. “ (personal communication by Keerti S. Rathore) Gene technology could improve food safety, food security and reduce environmental impact. Regulation is a major obstacle

  26. Lathyrus sativus A hardy tropical/subtropical legume Important source of nutrition but contains a neurotoxin: oxalyldiamino-propionic acid (ODAP) http://www.treknature.com/gallery/Asia/India/photo152618.htm

  27. http://www.gudjons.com/Mittel/Lathyrus-tub.jpg http://www.grainlegumes.com/fckeditor/aepfiles/File/Species/Lathyrus_sativus_pod_(L.delaRosa)_600.jpg • Beans from this so-called “famine crop” (consumed by poor people in Asia and Africa) causes lathyrism. • a paralytic disease (spastic paraparesis) prevalent among adults in Central India who have consumed large quantities of L. sativus seeds for several months • Safe content for ODAP is < 0.2%. Content in varieties range: 0.30-3.3 • Classical breeding approaches are in progress, but what about a transgenic approach targeting the toxin biosynthetic pathway only in the seed?

  28. Biosynthesis Proposed biosynthetic pathway for β-1 in Lathyrus sativus (products in brackets have not been detected) Yan et al., (2006) Phytochemistry 67:107–121

  29. Fonio & pearl millet cause goiter Gressel (2008) Genetic Glass Ceilings

  30. Courtesy of K. Petroni Courtesy of T. Maggiore Mycotoxins • Mycotoxins in grains are a major health problem (fumonisins and aflatoxins) • Cause cancer and neural tube defects  Improve resistance or increase degradation - direct: improve resistance to fungi (corn expressing plant defensin in field trials with encouraging results) - Engineer mycotoxin degrading activities - indirect: reduce insect damage through Bt toxin (effective for fumonisins)

  31. Courtesy of K. Petroni Tonelli, Pilu, Petroni (University of Milan, IT) Classical breeding (Flavonoid and anthocyanins) Some colored lines show lower levels of fumonisin compared to control yellow lines Transgenesis (Bt maize)

  32. Other examples by classical breeding Erucic acid in Brassica napus Cyanogenic glucosides in trifolium Glucosinolates in brassica • Many food security/safety problems in the developing world are waiting for a solution. Genetic manipulation is • more precise, more predictable, low cost solutions (not necessarily alternative to breeding) Consequences of toxin reduction

  33. Reducing glucosinolates in Arabidopsis • Glucosinolates are sulphur rich compounds from brassicas • Some beneficial, other toxic (quantity!) • Upon wounding are converted into toxic products • Regulators identified (two branches) • Mutants isolated

  34. Short chain Aliphatic GSL Long chain Indolic GSL Beekwilder et al., (2008) PLoS 3:e2068.

  35. Mutating Myb28 and Myb29 Regulators Beekwilder et al., (2008) PLoS 3:e2068.

  36. Reducing glucosinolate content... ...stimulates pest growth and damage! Beekwilder et al., (2008) PLoS 3:e2068

  37. A. thaliana making cyanogenic glucosides Effect on flea beetle and larvae feeding A) Adult beetles fed extensively only on leaves containing no dhurrin. B) Larvae frequently initiated no mines on leaves containing dhurrin, although attempts were made to feed (indicated by circles) Reduction in toxin content is a trade-off process: increases susceptibility to pests Tattersall et al., (2001) Science 293:1826-8

  38. Life is full of trade-offs • Reduction in pesticide content (a corollary of crop domestication) is not without consequences! • General pesticides (e.g. cyanide) are worse for humans than specific ones • Eat the pesticides you prefer (natural does not imply safer) Take home message (II)

  39. Allergens • Widespread occurrence • You may not know it until you experience it • Nuisance / cost / deadly threat • Minute amounts of allergens may cause a life-threatening anaphylactic reaction • May occur after ingestion, skin contact, injection of an allergen or inhalation. • 48 deaths caused by food over a 7-year period between 1999 and 2006 in UK Anaphylactic shock: when food kills www.youtube.com/watch?v=XC0nHFblLcE

  40. Allergies caused by plants Eight foods account for 90% of all food-allergic reactions. milk, egg, fish, shellfish, peanut, tree nut, soy, wheat Pollen is the major cause of respiratory allergy. At least 40% of type 1 allergic patients are sensitized against grass pollen allergens • Contrary to common perception, transgenic plants never caused allergic reactions to consumers. Many conventional crops do it regularly • If a gene used for transgenesis comes from a plant containing allergens, the transgene is checked for allergenicity

  41. Reducing plant allergens Transgenesis, rather than a cause of allergy, can be part of the solution • Apple • Peanut • Wheat (celiac disease) • Soybean • Ryegrass • Birch

  42. Soybean allergen: P34 US/Europe: 5 - 8% of babies and 2% of adults allergic* to soybeans Dominant soybean allergens is P34: > 65% of soy-sensitive patients react only to P34 protein Transgenic soybean without P34 published in 2003 (Herman et al., 2003) Herman et al., (2003) Plant Physiol. 132:36-43 No difference in composition, development, structure, or ultrastructure when compared with control plants. No other significant changes in polypeptide pattern

  43. “Regulatory difficulties and the lack of acceptance of GM soybeans by the baby food and formula industry makes using such an allergen-suppressed soybean difficult [read impossible] at the present time.” Alternative approach: identify soybeans with little/no allergen  screen the entire USDA national soybean germplasm collection Joseph et al. (2006) 46:1755-63 Out of > 16 266 accessions soybean germplasm screened, 12 lines (2 in the cultivated soybean) have no P34 allergen Why these two soybean plants lack the antigen?

  44. Where logic ends, biotech regulation begins If a protein is > 50% identical to an allergenic protein, it is a “potentially allergen”. Transgenic products have to be labelled Phaseolin ► eaten by one billion people everyday ► NOT recognized as an allergen BUT ► 54% similar to conglycinin (a minor soybean allergen) ► ‘potential allergen’ according to biosafety regulation Transgenic Cassava expressing phasolin produced by C. Faquet (Danforth) * improved (350%!) protein content * freely available (no royalties) in developing countries but phaseolin similarity to a know allergen is 54% (>50%)  transgenic cassava would require labelling (obviously impossible & ridiculous) in Africa

  45. Two texts with 60% similarity The glory of Him who moveth everything Doth penetrate the universe, and shine In one part more and in another less (Dante, Paradise, Canto I, v.1-3) The story of him who believeth everything Does perpetuate diverse lies and causes one part of farmers or another to die 60 % similarity does not give the same effect New almond or peach varieties may accumulate much more cyanogenic glucosides, new potato varieties may accumulate more or new glycoalkaloids. In Italy and the EU they require no regulatory scrutiny (no compulsory tests) before release, cultivation or commercialization if they are produced by conventional breeding or mutagenesis.

  46. Conclusions • Plant derived allergens and toxins are ubiquitous, abundant • Tools are available to reduce them (conventional or transgenic) • Strategies must be reasonable (accept some level of risk) [risks / benefits] • Overcautious regulation kills the technology and associated benefits

  47. Time to say the truth A man came home at noon, said good night to everybody, and went to bed.  His wife, much concerned, asked him if he were ill, and the good man answered:  'I am quite well.  But this morning everybody told me that I was drunk.  I am not drunk -- you know that I do not even take wine, that I am an abstainer -- but I love peace, and in order not to contradict them I am going to bed.'  Plant biotechnologists avoided discussions (complied to insane regulations) for the sake of peace. Time has come to raise the voice and demand a change Present regulation is unscientific, very costly, excessive, restricting the potential uses of the technology to few crops and is aborting the technology in and for developing countries. Propagation of unreasonable and unfounded fears about biotechnology mantains a high regulatory burden and keeps de facto the technology in the hand of few industrial groups which are interested only in few crops/problems

  48. The P38 and the apple A true desire, when does not come to terms with reality, but follows the path of irrational utopia, turns into lie and can only end up in murderous madness and self destruction. S. Allevato e P. Cerocchi (2009) “La P38 e la mela”, publ. ITACA, p.173 Un desiderio vero, quando non fa i conti con la realtà, ma imbocca la strada dell’utopia irrazionale, diventa menzogna, e non può che condurre alla follia omicida e all’autodistruzione. S. Allevato e P. Cerocchi (2009) “La P38 e la mela”, ed. ITACA, p.173

  49. Acknowledgements • Support or material from colleagues and friends (especially Parrot, Gressel Kershen, Salamini, Fico, Vitale, Ederle, Maggiore, Petroni, Rossi…) • Confidential information or help with literature (Gilissen, Bisht, Rathore, Shah, Carputo, Parisi, Faquet …) • The support of the CSBA (access to specific books) and Univ. of Milan

  50. Bibliography silencing/mutations • Carlini and Grossi-de-Sa´ (2002) Toxicon 40:1515–1539 • R Koes et al. (1995) Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants. Proc Natl Acad Sci U S A. 92:8149-8153. • Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: 950–952 • Batista • Napoli et al. (1990) Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 2:279-289 • van der Kroll et al. (1988) An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation Nature 333:866-869

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