Research Project:

The Story of the American

Chestnuts

Castanea dentata: “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

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

• 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

How the Blight Works

~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

Transgenic Trees & Synthetic Biology

• 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

•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

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

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.

Current “Management”

clothianidin

thiamethoxamimidacloprid

Design Proposal

Engineer a “smart” bacteria with the ability to:

• Locate P. stewartii• Destroy the bacterial colony• Be non-pathogenic to humans, animals,

corn• Essentially function as an

environmentally friendly, pathogen-specific pesticide

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.

Bdellovibrio bacteriovorus

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

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.

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

Detection of OHHL by B.

bacteriovorus

Positive Chemotaxis

Transcription of MotAB3

Mean Swim Speed (μm/s)

+/- SD*

0 0 0 26.5 +/- 1.8

1 1 1 63.2 +/- 5.5

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*

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.

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