Engineered herbicide resistance in crop plants

1. Engineered herbicide resistance in crop plants Presented by Mark Horsman February 16th, 2012

2. Outline • Focus of talk • Herbicide usage levels • Classes of herbicides • Imidazolinones targeting acetolactate synthase • Glusulfinate targeting glutamine synthetase • Glyphosate targeting 5-enolpyruvoyl-shikimate-3-phosphate synthetase • Marketing resistant crops and discussions of GURT

3. Treatments for plants • Microbicides/Fungicides • Insecticides • Bacillus thuringiensisCry toxin expressed in-plant • Hormones • Herbicides • Defoliants • Selective weeding tool

4. Herbicides • Treatment of field with microbes and fungae • Plant-expressed chemicals (e.g. Juglone from Black walnut) • Chemical interference with photosynthesis, growth regulation, and metabolism • The herbicide must be effective against problem plants without damaging the crop of interest

5. Methods of herbicide selectivity • Target differences in growing times • Take advantage of differences in absorption and metabolism. Weeds and crop fall under two main classes: • Monocots (grasses) • Dicots (broad leaf) • Use crops with acquired resistance to non-selective herbicides

6. Plants by class Photo credited to Peter Halasz

7. Acquiring resistance to herbicides • Breeding • Mutant screening • Genetic Engineering Load gene into carrier for transformation into plant Transform gene into plant tissue and grow into full plant Isolated and modify gene of interest

8. Science Magazine, April 2008 online feature

9. Genetically modified crop (corn) adoption

12. Major classes of acquired-resistance herbicides

13. Major classes of acquired-resistance herbicides

14. Outline • Focus of talk • Herbicide usage levels • Classes of herbicides • Imidazolinones targeting acetolactate synthase • Glusulfinate targeting glutamine synthetase • Glyphosate targeting 5-enolpyruvoyl-shikimate-3-phosphate synthetase • Marketing resistant crops and discussions of GURT

15. Branched-chain amino acid synthesis and the acetohydroxy acid intermediate

16. Aceto-hydroxyacid synthesis via thiamine pyrophosphate

17. (McCourt, Pang et al. 2006)

18. ALS-mediated activation of TPP

19. ALS-mediated activation of TPP

20. (McCourt, Pang et al. 2006)

21. Acetolactate Synthase PDB 1Z8N(McCourt, Pang et al. 2006) α Domain β Domain γ Domain

22. Proximity of the two rings

23. Forced V-shape

24. Imazaquin

25. Related Imidazolinones

26. Adapted from Duggleby, R. G. et al. 2008

27. Sites of mutation Adapted from Duggleby, R. G. et al. 2008

29. Relative AHAS inhibition kinetics Kawai et al. (2008) found that • Rice AHAS shows an imazaquin IC50 of 2.2 µM • A tryptophan 548 to leucine, serine 627 to isoleucine double mutant displayed 12% inactivation at 100 µM

30. Result of non active-site mutations • Mutant AHAS/ALS genes with altered residues at arginine 377 and tryptophan 574 confer resistance crop resistance to sulfonylureas and imidazolinones without reducing catalytic activity • These mutations have been observed naturally in several grasses (including varieties of corn). These herbicides are more effective against dicots.

31. Outline • Focus of talk • Herbicide usage levels • Classes of herbicides • Imidazolinones targeting acetolactate synthase • Glusulfinate targeting glutamine synthetase • Glyphosate targeting 5-enolpyruvoyl-shikimate-3-phosphate synthetase • Marketing resistant crops and discussions of GURT

32. Plant Glutamine Synthetase • Ammonia incorporation method in plants • Disruption causes ammonia poisoning

34. Glutamate homologues

35. Unnoet al.’s 2006 crystal structure of a maize glutamine synthetase PDB 2D3A

36. PDB 2D3B

37. PDB 2D3C

38. PDB 2D3C

39. Resistance acquired throughPhosphoinothricin acetyl-transferase Acetylation removes hydrogen-bonding with glutamic acid 132 and prevents PPT from docking in the active site

40. Outline • Focus of talk • Herbicide usage levels • Classes of herbicides • Imidazolinones targeting acetolactate synthase • Glusulfinate targeting glutamine synthetase • Glyphosate targeting 5-enolpyruvoyl-shikimate-3-phosphate synthetase • Marketing resistant crops and discussions of GURT

41. Aromatic amino acid Biosynthesis Adapted from Bongaerts, J., M. Kramer, et al. (2001)

42. Shikimate pathway inhibition Adapted from Funke (2006)

44. Type I EPSPS stabilizing glyphosate Schonbrunn 2001 PDB 1G6S

45. Comparing Type 1 and Type 2 EPSPS’s Sequence identity is 29% PDB 1G6S PDB 2GGA

46. Glyphosate in the native Type II EPSPS active site PDB 2GGA

47. Funke (2006)

48. Mutated (A100Q) Type II EPSPS active site PDB 2GGD

49. Agrobacterium tumefaciens type 2 EPSPS maintains catalytic activity • The differences in overall protein structure allow type 2 EPSPs to selectively exclude glyphosate while still reacting with PEP

50. Comparing kinetic parameters 1Funke (2006), 2 Eschenburg (2002)