BBMB 607 September 10, 2010 Presented by Matt Crispin

1. Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insectsKandoth, K.P., Ranf, S., Pancholi, S. S., Jayanty, S., Walla, M. D., Miller, W., Howe, G.A., Lincoln, D.E., Stratmann, J.W. 2007. PNAS USA, Vol 104, pg 12205-12210 BBMB 607 September 10, 2010 Presented by Matt Crispin

2. Connection To My Work • Plants can defend using specialized metabolites. • Specialized insects do more damage to H. perforatum leaves but cause less defensive response than a generalist. (Sirvent, 2003). • Methyl jasmonate can cause increases in alkamides which are defense compounds in E. pallida (Binns, 2000) Amide 1 Hyperforin Hypericin

3. Background • Systemin deficient mutants have lower resistance to M. sexta larva, systeminconstituitive have higher. • Perception of systemin by SR160 creates many responses early responses including JA synthesis. • MAPKs are not yet known to function in the systemin and wounding inducing signaling pathway. • MAPKs are part of a three-tiered phosphorelay cascade.

4. Background For LeMPK 1, 2, and 3 • LeMPK1 and 2 are 95% identical at the amino acid level. • Activity is upstream to JA biosynthesis. • Phosphorylated post translationally, not upregulated by systeminor wounding. • LeMPK3 is transcriptionallyupregulated by wounding. • One of the few substrates for these are 1-aminocyclopropane-1-carboxylate synthases 2 and 6. • All are activated by AvrPto and AvrProB from P. syringae.

5. Materials and Methods • Creation of Virus Induced Gene Silencing constructs • Creation of transgenic tomato • MAPK and PI-II Assays- In gel kinase assay with myelin basic protein as an artificial substrate. MPK 1 and 2 have manganese and MPK 3 has magnesium in kinase buffer. • JA analysis-Used GC-MS with dihydro-JA internal standard • MeJA treatments • Tissue analysis • Herbivore Treatments

6. VIGS

7. Specific Aims • To test if silencing LeMP1 and 2 reduces activation of systemin induced late genes, and JA biosynthesis. • Test if silencing any one of the three has a similar effect to cosilencing. • Test if silencing reducing resistance to herbivores.

8. Cosilencing of 1/2 • Cosilencing lessens 1/2 protein amount, mRNA amounts, and activity. • Lowers PI-II as well. • MPK3 not affected.

9. Effects of MPK 1/2 on Late genes

10. Effects of silencing on JA and PI-II

11. Silencing of LeMPK 1, 2, and 3 individually

12. Activity after individual silencing

13. Effect of Silencing MPK3

14. M. sexta size changes between control and VIGS plants

15. Conclusions • All three of the MPKs are essential to systemin induced wound signaling. • These MAPKs could be upstream or downstream of JA synthesis. • Late genes could be restored with MeJA. • One of two of LeMPK 1 and 2 is not sufficient for late gene activation. • MPK 3 has a different action than 1 and 2.

16. Future Experiments • Testing how MAPK can create different responses to different stresses. • I would like to see the effect of using specialized compared to generalized feeders this whole system.

17. Literature Cited • Gibson, D.M., Krasnoff, S.B., Sirvent, T.M. 2003. Induction of Hypericins and Hyperforins in Hypericum perforatum in Response To Damage By Herbivores. Journal of Chemical Ecology Vol 29 12:2667-81 • Binns, S.E., Inparajah, I., Baum, B.R., Arnason, J.T. 2000. Methyl jasmonate increases reported alkamides and ketoalkene/ynes in Echinacea pallida (Asteraceae). Phytochemistry 57:417-20 • Waterhouse, P.M., Helliwell, C.A. 2003. Exploring plant genomes by RNA-induced gene silencing. Nature Reviews Genetics, Vol 4: 29-38