Heather Hallen spent eight years looking for poison in all the wrong
places. Alpha-amanitin is the poison of the death cap mushroom, Amanita
phalloides. The Michigan State University plant biology research associate
was looking for a big gene that makes a big enzyme that produces
alpha-amanitin, since that's how other fungi produce similar compounds.
But after years of defeat, she and her team called in the big guns -
new technology that sequences DNA about as fast as a death cap mushroom can
kill.

The results: The discovery of remarkably small genes that produce the
toxin - a unique pathway previously unknown in fungi.

The discovery is reported in today's Proceedings of the National
Academy of Sciences. It is work that not only solves a mystery of how some
mushrooms make the toxin - but also sheds light on the underlying
biochemical machinery. It might be possible one day to harness the mushroom
genes to make novel chemicals that would be useful as new drugs.

"We think we have a factory that spits out lots of little sequences to
make chemicals in Amanita mushrooms," said Jonathan Walton, MSU plant
biology professor who leads Hallen's team. "Our work indicates that these
mushrooms have evolved a mechanism to make dozens or even hundreds of new,
previously unknown chemicals, besides the toxins that we know about."

Of the thousands of species of mushrooms, only about 30 produce
alpha-amanitin.

Most of them look much like their edible cousins. But poisonous
mushrooms are powerful in folklore and in history. In 54 A.D., Emperor
Tiberius Claudius was fed a death cap mushroom by his wife Agrippina to put
her son Nero on the throne of Rome.

Alpha-amanitin kills people by inhibiting an enzyme necessary for
expression of most genes. Without the ability to synthesize new proteins,
cells quickly grind to a halt. The intestinal tract and the liver are the
hardest hit as they come into first contact with the toxin. By the time
symptoms show up, a liver transplant is often the only hope.

Hallen, a mycologist, gathers mushrooms in the Michigan woods and
often is called upon to help identify mushroom species for veterinarians,
parents of small children and local hospitals - often in a desperate race to
beat alpha-amanitin's effects.

Walton's lab works to understand the biochemical pathways by which
natural products are synthesized in fungi. Fungal natural products that
benefit human health include penicillin and the immunosuppressant drug
cyclosporin.

Studying their biosynthesis could lead to the discovery and
development of new medicines.

To find the elusive gene for alpha-amanitin, they used what they term
"brute force" - a new machine at MSU that can sequence immense quantities of
DNA quickly. The 454 LifeSciences pyrosequencer generates 100 Mb DNA
sequence in one overnight run - twice the size of a fungal genome.
Traditional sequencing methods require months to yield the same quantities.
What they found was a gene that encodes the toxin directly - with no need to
first synthesize an enzyme that in turn would make the toxin.

"The RNA goes in, and out comes the backbone of the toxin," Hallen
said. After its initial synthesis, the toxin is then modified in several
ways by the mushroom to make it exceptionally poisonous.

Walton said the discovery poses some interesting evolutionary
questions. For example, why do only some mushrooms produce this toxin" And
how did a handful of other, unrelated mushrooms evolve the same trait"
Finding the genes points to how the trait could appear in one mushroom, but
not how it evolved in mushrooms that aren't related to Amanita.

Hallen and Walton also see the doors opening to a diagnostic test that
could use DNA to determine if a mushroom is toxic or not. Identifying a
mushroom by shape and color alone is often impossible if the mushroom has
been cooked or partially digested, yet rapid and accurate identification in
an emergency room situation is critical.


[www.msu.edu]