Research Project: The Story of the American Chestnuts
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
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
~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, 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
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.
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.
Bdellovibrio bacteriovorus B. Bacteriovorus is a predatory Gram-negative bacteria that preys on other Gram-negative bacteria.
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.
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
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. * 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.