1. Plant Volatiles – Chemical Defense Mechanisms Symbiotic, antibiotic, and defense relationships Acacias – sugar composition adjusted to desired ant species Heil et al. (2005) Postsecretory hydrolysis of nectar sucrose and specialization in ant/plant mutualism. Science 308 (5721) Plants provide sugars for which ants have no catabolic enzyme.

2. predator’s Plant predator predator Plant-Herbivore-parasiticInsect “Tri-trophic” Interactions

3. Schnee et al. (2006) The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. PNAS 103, 1129 “Tri-trophic” Interactions maize, cotton, etc. e.g. Spodoptera littoralis parasitic wasps feeding damage forced regurgitating

4. JA biosynthesis – abbreviated VOC – volatile organic compounds From plant signaling to insect response via Farmer & Ryan (early 90s) – volatile signals from plant to plant Jasmonates Terpenes

5. Plants respond to caterpillar feeding Turlings TCJ, Loughrin JH, McCall PJ, Rose USR, Lewis WJ, Tumlinson JH (1992) How caterpillar- damaged plants protect themselves by attracting parasitic wasps. PNAS 92, 4169. Healthy, undamaged corn seedlings 6 hours after start of caterpillar feeding IS1,2 – internal standards Some peak IDs (LC-MS): 1,2,3 – 3-hexenal; 2-hexenal; 3-hexenol 5- linalool; 9 – β-farnesene; 10 - nerolidol C6 C10 C15 10 9 5 1 C15

6. jasmone indole Feeding on cotton 1 st day 3 rd day linalool pinene farnesene Change in composition over time of attack. Signaling compounds (or degradation products) at low levels.

7. Emitted compounds by cotton Start - 2 p.m. 5 caterpillars on 6w-old cotton A – LOX products from cotton B – constitutive cotton volatiles C – induced compounds in cotton

8. Emissions by infected corn over time LOX-products from corn Induced complex compounds Leaves scratched, then added caterpillar regurgitate Recognition – timing, composition and nature of compounds

9. Signals in caterpillar “spit” induceplantbiodefenseWMD by recruiting allied forces Based on Isoprene & Isoprenoid metabolism acetoacetyl-CoA + acetyl-CoA > HMG-CoA > mevalonate >>>> isopentenyl-PP C4 + C2 > C6 > C5 + CO 2

10. Isoprene Isopentenyl-PP Dimethylallyl-PP Geranyl-PP C5

11. C20 - Geranyl-geranyl-PP C15 – farnesyl-PP C25 – Sesterterpines > abundant, non-volatile C30 - Triterpenes > steroid source structure, abundant, non-volatile C40 - Carotenes > carotenoid source structure, abundant, non-volatile 6β-acetoxy-24-methyl- 12, 24-dioxoscalaran-25-al (pacific sponge) Sesquiterpene type – phytol (retinol, retinal) Cyclic sesq. (cadinene)

12. Induction of sesquiterpene synthases Wasps fly straight to damaged leaf from downwind, not to a wounded leaf, but to wounded leaves treated with regurgitated midgut sap of insect. maize

13. bergamotene farnesene sesquiphellandrene Gene to Product maize

14. What happens when the gene is expressed in Arabidopsis ? A single transgene/ protein generates the entire spectrum! … but will the wasps know?

16. Wt and transformed Arabidopsis – wasps in central compartment wt tr P < 0.01 naïve wasps trained on Arabidopsis trained on maize Side result – wasps must learn by trial & error, i.e., there are other cues; signals that connect wasp & caterpillar

17. One could use the contraption for other experiments Western Corn rootworm Diobrotica v. virgifera A major problem in US agriculture – is there a natural biodefense strategy (i.e., no chemicals)? Metabolomics to the Rescue!

18. One could use the contraption for other experiments Maize Western Corn rootworm Nematode Trimorphic interaction involving a entomopathogenic nematode Rasmann et al. (2005) Nature 434, 731.

19. Experiments similar to the wasp predation experiment Identification of attractant Why is US maize not protected Does it work in the field Isoprenoids in the soil?

25. Sesquiterpene hydrocarbons in maize A – leaf inducible, B – ubiquitous; C – root specific

26. Terpene synthases in maize Heterologous expression GC-MS with isotopic tracers GC-MS of different lines Mutational analysis Sesquiterpene spectrum as affected by mutational analysis of the gene