Research Project: The Story of the American Chestnuts

1. Research Project: The Story of the American Chestnuts

2. Castaneadentata: “Redwoods of the East” • Native to New England and the Appalachian Mts. • 1 of every 4 trees in old-growth Appalachian forests was an American chestnut. • 100+ feet tall, 10 ft. diameter trunks • Gave food & shelter to black bears, wild turkeys, Carolina parakeets, moose, elk, deer, mountain lions… • Rot- and water-resistant wood that split easily • Abundant annual nuts used in traditional American recipes • Used for railroads, instruments, housing, telegraph/telephone poles, etc. • Tannin extracts used for tanning leather • Timber an important export to the early American economy

4. Blight Introduced from Asian Chestnut Varieties

5. How the Blight Works • Cryphonectriaparaciticaenters through a wound, colonizes beneath bark • Spreading hyphae produce oxalic acid and kill the cambium, an vital layer of bark • Disease spreads up tree, but not to roots • Roots attempt to regrow • Result: “living stumps” that never grow tall

6. ~1980-2013 : The Backbreding Method • Project led by The American Chestnut Foundation (TACF) • Utilizes Mendelian genetics • First use of “gene knockout” on trees • Goal: the “perfect” hybrid

8. Transgenic Trees & Synthetic Biology • March 2013: OxO wheat gene and strong promoter, CaMV35S inserted into American chestnuts • April 2013: Powell’s transgenic trees planted in New York where the first trees died in 1910, hope for recovery • Hybrid tree development time-consuming, still haven’t developed a flawless tree • William Powell from SUNY-ESF begins developing GM trees as an alternative • Realized Triticumaestivum, common wheat, has genes for proteins that break down oxalic acid

10. Design Project:N-Acyl Homoserine Lactone (N-AHL) Directed Bdellovibro bacteriovorus

11. Stewart’s Wilt (P. stewartii) Stewart’s wilt is a corn disease caused by a rod-shaped, Gram-negative bacteria. Corn flea beetles bring the bacteria into crops every Spring. P. stewartii overwinters in the beetle’s gut every year.

12. Current “Management”

13. Design Proposal

14. Quorum-Sensing in Stewart’s Wilt The Gram-negative quorum sensing systems are all very similar to the two-component system used by vibrio fischeri, the first discovered quorum-sensing system. But, the QS proteins EsaI and EsaR in P. stewartii are slightly different: • EsaI creates the N-Acyl homoserine lactone OHHL • EsaR binds to OHHL, and detatches from the promoter it is otherwise bound to Thus, EsaR is unique in that it acts as a repressor, and AHL molecules cause derepression instead of the usual activation caused by Quorum sensing receptor proteins.

15. Bdellovibrio bacteriovorus B. Bacteriovorus is a predatory Gram-negative bacteria that preys on other Gram-negative bacteria.

17. MotABprotein pairs are transmembrane protein complexes which affect flagellarrotation. B. bacteriovorus HD100 has three of these protein pairs that make up a hybrid motor. Each protein pair contributes to rotational power, but MotAB3 has the most significant impact.

18. The chemotactic system utilizes methyl-accepting chemotactic proteins (MCPs), transmembrane proteins that detect and bind to external ligands, in order to set off a chain reaction of proteins in the chemotactic system. • These reactions then cause a “biased random walk” towards the chemical detected by the MCP. Traits of a biased random walk: • Increased likelihood of “tumbling” when moving down a concentration gradient • Prolonged “smooth swimming” when moving up a concentration gradient

19. Design Proposal, revisited • Add an esaR gene sequence to B. bacteriovorus such that an EsaR protein sits on the MotAB3 operon promoter. • Artificially construct an MCP that accepts OHHL as its ligand. Replace all naturally occurring MCPs. * http://jb.asm.org/content/193/4/932.full

20. Ideal Outcome • These two alterations of B. bacteriovorus are regulated by the same external chemical, OHHL. • Ideally, this concurrent swim speed increase and directional bias will “lock in” B. bacteriovorus to the desired prey, thus causing a dramatic increase in the probability of prey being P. stewartii. • If predation levels are adequate, B. bacteriovorus will be able to repress concentrations of the pathogen and prevent severe cases of Stewart’s wilt in corn.

21. Considerations, Good and Bad