1. Research Project:
The Story of the American Chestnuts

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

3. Blight Introduced from Asian Chestnut Varieties
1870: Imported Chinese and Japanese chestnut trees, better for orchards and nut harvesting.
1904: Blight detected in American chestnuts
1910: American chestnuts at the Bronx Zoo die
1940 – 1960: Nation-wide efforts to stop blight spread, breed Asian-American hybrid
1960: 4 billion American chestnuts lost, U.S. Gov’t ceases funding for restoration efforts

4. How the Blight Works
Cryphonectria paracitica enters 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

5. ~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

7. Transgenic Trees & Synthetic Biology
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 Triticum aestivum, common wheat, has genes for proteins that break down oxalic acid
March 2013: OxO wheat gene and strong promoter, CaMV 35S inserted into American chestnuts
April 2013: Powell’s transgenic trees planted in New York where the first trees died in 1910, hope for recovery

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

10. 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.

11. Current “Management”
clothianidin
thiamethoxam
imidacloprid

12. 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, which we discussed in class.
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 activation.

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

15. MotAB protein pairs are transmembrane protein complexes which affect flagellar rotation. 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.

16. 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

17. Design Proposal
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.
Detection of OHHL by B. bacteriovorus
Positive Chemotaxis
Transcription of MotAB3
Mean Swim Speed (μm/s) +/- SD*
26.5 +/- 1.8 1
63.2 +/- 5.5 *

18. 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.

19. Considerations, Good and Bad
Positive
Could replace harmful pesticides that otherwise damage the ecosystem
Safe and easy to test (disregarding the development hurdle)
Potential model for targeting other Gram-negative bacteria
Negative
Increased probability of predation does not guarantee P. stewartii as the sole prey
P. stewartii is not detected until harmful levels are already present within the plant
More research is required to understand the structure and function of MCPs