The Amanita Genome Project


Heather Hallen, Ph.D.
Research Associate, MSU DOE Plant Research Laboratory, 106 Plant Biology Laboratories, Michigan State University, East Lansing, MI 48824-1312, USA. hallenhe@msu.edu

Jonathan Walton, Ph.D.
Professor, MSU DOE Plant Research Laboratory, 106 Plant Biology Laboratories, Michigan State University, East Lansing, MI 48824-1312, USA. walton@msu.edu

 

For the most up-to-date information on the Amanita genome project, go to: http://www.msu.edu/user/hallenhe/

 

What is Amanita? Why a genome project?

Amanita bisporigera is a hymenomycete fungus endemic in North America (Fig. 1). Amanita species, including A. bisporigera, form ectomycorrhizae, obligate mutualistic associations with forest trees. To date, no mycorrhizal fungus has had its genome sequenced (Laccaria bicolor is in the process of being sequenced by the DOE Joint Genome Institute, with completion projected for 2005). The database of the International Ectomycorrhiza Genome Project <http://mycor.nancy.inra.fr/ectomycorrhizadb/> currently contains 5685 ESTs, from three organisms (basidiomycetes Laccaria bicolor and Pisolithus microcarpus, and ascomycete Tuber borchii), with links to an additional 13,596 ESTs and genomic sequences, representing Heboloma cylindrosporium and our own Amanita. As the ectomycorrhizal symbiosis is crucial for the health of forest ecosystems worldwide and the associated forest products-based economies, the genes and genetic controls associated with mycorrhizae are of great interest worldwide.  
  

Amainta, with yellow stain

Several specimens of A. bisporigera

Fig. 1. Fruiting body of  Amanita bisporigera, showing yellowing in response to KOH (Photo by H. Hallen)

Fig. 2. Several specimens of Amanita bisporigera, showing partial veil (annulus) and universal veil (volva). (Photo by H. Hallen)

        

Amanita bisporigera and its close relatives in genus Amanita, section Phalloideae are responsible for 90% of fatal human mushroom poisonings. Among several secondary metabolites of importance, A. bisporigera produces amatoxins and phallotoxins (Fig. 2). Amatoxins, including a- and b-amanitin  (commercially available), are specific inhibitors of eukaryotic RNA polymerase II and are thus important molecular biological tools in addition to being potent natural toxins. Functional amatoxins cannot be artificially synthesized (Wieland & Faulstich 1991). Phallotoxins also play a role in the molecular biology laboratory, as they bind to actin and stabilize it in the filamentous F-actin form.

 

Amatoxins and phallotoxins are cyclic peptides and share several structural similarities (see Fig. 2). Because of the important roles these chemicals play in molecular biology and, in the case of amatoxins, in vertebrate poisonings, elucidating the biosynthetic pathways is of considerable interest. Based on similarities to other fungal secondary metabolites, amatoxins and phallotoxins are likely to be possess a non-ribosomal synthesis pathway (Walton et al. 2004). This would imply very large, clustered biosynthetic genes (approximately 30 kb for the putative amatoxin synthetase gene, 27 kb for phallotoxin synthetase). Non-ribosomal peptide synthetases share numerous conserved motifs that should be readily apparent in a relatively crude genome sequencing project. For a variety of reasons, other approaches towards characterizing these genes (PCR- and reverse transcript PCR-based methods; ATP-pyrophosphate exchange assays) have been unsuccessful. Amanita species neither produce toxins nor fruit in culture. All in vivo toxin synthesis appears to take place during a small window (likely twelve hours or less) at or near the time of fruiting body initiation, while the fungus is still subterranean (Preston et al. 1982; pers. obs.).


Figure 2. Structural formulae for common amatoxins and phallotoxins.

amatoxin structure

Distribution:

Amatoxins are produced by three genera of fungi in addition to Amanita: Galerina, Lepiota and Conocybe (Benjamin 1995). Phallotoxins are produced by Conocybe as well as by Amanita species (Hallen et al. 2003). Many other fungal secondary metabolites share similarly disjunctive distributions, and horizontal transfer has been proposed as a controversial hypothesis to account for this (Walton 2000). To date, horizontal transfer of secondary metabolite clusters in fungi has been neither proven nor disproven. Obtaining amatoxin or phallotoxin biosynthetic gene sequence from Amanita would allow us to determine the gene structure in the other toxin-producing genera. By comparing a toxin biosynthesis gene tree with extant, rRNA gene-based species trees (Hallen 2002; Hallen et al. 2003; Moncalvo et al. 2002), we could readily test the hypothesis of horizontal transfer, obtaining a conclusive answer one way or the other for this group of organisms and metabolites.


Progress to date:


We have generated 9560 unidirectional sequencing reads, amounting to approximately 5.7 Mb or about 14% of the total genome.

 

We presented both a talk and a poster at the 23rd Fungal Genetics meeting in Asilomar, March 15-20, 2005. A pdf version of the poster is available here.

 

This work has been funded by a grant from the U.S. Department of Energy to the Plant Research Lab, Michigan State University. For information including access to the sequence data, contact either Heather Hallen (hallenhe@msu.edu) or Jonathan Walton (walton@msu.edu).

 

This web page is maintained for general interest only. For the most up-to-date information on the Amanita genome project, go to: http://www.msu.edu/user/hallenhe/

 

Links of interest

<http://pluto.njcc.com/~ret/amanita/mainaman.html> - Your best source for All Things Amanita, maintained by Dr. Rodham Tulloss and Dr. Zhu-Liang Yang.
<http://www.amanitabear.com> - similarity calculator, mycological consulting and more (hard-core mycological tools)

<http://www.basidiomycetes.org> - A gateway to basidiomycete research, including basidiomycete genome projects.

<http://wit.integratedgenomics.com/GOLD/> - Genomes On Line Database. Summary of publicly funded genome projects.
<http://www.msafungi.org/> - Mycological Society of America website.

<http://www.tomvolkfungi.net> - Home of the Fungus of the Month, among other fascinations.
</> - The Walton Lab webpage. Amanita's not your thing? - Then check out the exciting world of host-specific toxins, Cochliobolus carbonum, maize cell walls and more!

<http://www.msu.edu/user/hallenhe/> - More fun with Amanita, amatoxins, general mycology, and the odd chameleon, at Heather Hallen's webpage.


References

Benjamin DR (1995) Mushrooms: Poisons and Panaceas. New York: WH Freeman and Company. 422 pp.

 

Birren B, G Fink, E Lander (2002) Fungal genome initiative: white paper developed by the fugal research community. Whitehead Institue Center for Genome Research, Cambridge, MA. <http://www.broad.mit.edu/annotation/fungi/fgi/FGI_01_whitepaper_2002.pdf>

 

Hallen HE (2002) Studies in amatoxin-producing genera of fungi: phylogenetics and toxin distribution. Ph.D. dissertation, East Lansing, Michigan: Michigan State University. 192 pp.

 

Hallen HE, R Watling, GC Adams (2003) Taxonomy and toxicity of Conocybe lactea and related species. Mycological Research 107:969-979.

 

Kelkar HS, J Griffith, ME Case, SF Covert, RD Hall, CH Keith, S Oliver, MJ Orbach, MS Sachs, JR Wagner, MJ Weise, JK Wunderlich, J Arnold (2001) The Neurospora crassa genome: cosmid libraries sorted by chromosome. Genetics 157:979-990.

 

Moncalvo JM, R Vilgalys, SA Redhead, JE Johnson, TY James, MC Aime, V Hofstetter, SJW Verduin, E Larsson, TJ Baroni, RG Thorn, S Jacobsson, H Clemencon, OK Miller, Jr. (2002) One hundred and seventeen clades of euagarics. Molecular Phylogenetics and Evolution 23:357–400.

 

Preston JF, BEC Johnson, M Little, T Romeo, HJ Stark, JE Mullersman (1982) Investigations on the function of amatoxins in Amanita species: a case for amatoxins as potential regulators of transcription. In: Peptide Antibiotics - Biosynthesis and Functions. H Kleinkauf & H von Döhren, eds. Berlin, Germany: Walter de Gruyter. pp. 399-426.

 

Walton JD (2000) Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: an hypothesis. Fungal Genetics and Biology 30:167-171.

 

Walton JD, DG Panaccione, HE Hallen (2004) Peptide synthesis without ribosomes. In: Advances in Fungal Biotechnology for Industry, Agriculture and Medicine. J. Tkacz and L. Lange, eds., Kluwer Academic, New York, pp. 127-162.

 

Wieland T, H Faulstich (1991) Fifty years of amanitin. Experimentia 47:1186-1193.

 

Zolan ME (1995) Chromosome-length polymorphism in fungi. Microbiol Rev 59:686-698.

 

 

 

updated 9/11/06 by JW