Genus Amanita

1. Gene family encoding the major toxins of lethal Amanita mushroomsorDude, where’s my nonribosomal peptide synthetase gene?

2. Genus Amanita Some of the most beautiful and some of the most lethal mushrooms in the world. Divided into several sections based on morphology and toxicity, including: Amanita Caesarea Phalloideae Vaginatae Validae

3. LSU phylogency of Agaricales Amanita is a relatively young genus and is divergent from its relatives in a number of ways. For example, amanitas do not produce the peroxidases found in other basidiomycetes. The activation domains in their endochitinases are different from other fungi and resemble those from lysozymes in animals. Source: Moncalvo et al. 1996. Molecular phylogeny of the Agaricales based on 25s rdna sequences. Asilomar Fungal Genetics Conference

4. LSU phylogeny of genus Amanita Figure 2:Strict consensus of 13 most parsimonious trees and phylogenetic classification of Amanita.  Numbers and letters above branches denote taxonomic groupings (clades) discussed in the text.  Synapomorphies in the phylogram are as follows: 1A spores not amyloid; 1B spores amyloid; 2A distinct basal bulb, volva not saccate; 2B no basal bulb; 3A annulus present;3B exannulate; 4A exannulate;4B annulus present, spores elongate; 5A ?; 5B appendiculate margin;6A volva not saccate, small spores;6B saccate volva; 7A spores globose; 7B volva friable; 8A inner layer of volva friable;8B volva entirely membranous. Source: Drehmel et al. 1999. Molecular phylogeny of Amanita based on large-subunit ribosomal DNA sequences: implications for taxonomy and character evolution. Mycologia 91(4):610-618

5. Section Validae(Amanita fulva) Section Caesareae(Amanita jacksoni) AND… Section Amanita(Amanita muscaria) Section Vaginatae(Amanita vaginata)

6. Section Phalloideae(the killers are in here) The death caps and destroying angel mushrooms produce the most lethal fungal toxins known. Amanita virosa Amanita ocreata Amanita phalloides

7. Amatoxins Alpha-amanitin is a powerful transcription inhibitor. It binds to RNA polymerase II, resulting in death in 36 to 72 hours.A few compounds may decrease uptake if administered immediately, but there is no antidote. A liver transplant is necessary to save someone who has ingested a death cap. In humans, LD50 is approximately 0.1 mg per kg of body weight. A single mushroom contains about 10 mg of the toxin — more than enough to kill a healthy adult.

8. Phallotoxins Another group of toxins produced by Phalloideae, these are potentially deadly but are not absorbed when ingested by mammals. They may act as protection against invertebrates, however. Phalloidin is known to bind to actin and prevent it from depolymerizing by interfering with ATP metabolism, effectively paralyzing cells. It is sometimes used as part of a fluorescent tag for staining. Human macrophage stained with Fluor488-phalloidin (green) (Source: ImproVision)

9. Cyclic Peptides in Fungi Amatoxins and phallotoxins are cyclic peptides. All such compounds are produced by pathways involving nonribosomal peptide synthetases (NRPSs) in other fungi. Predicted module arrangement of the A. fumigatusNRP synthetase gene (pesM), a typical fungal NRPS gene, showing three adenylation (A), three thiolation (T) and three condensation (C) domains, in addition to one epimerase (E) and one thioesterase (TE) domain. (Stack 2007) Source: Stack D et al, 2007. Nonribosomal peptide synthesis in Aspergillus fumigatus and other fungi. Microbiology 153: 1297-1306

10. Dude, where’s my NRPS gene? The Phalloideae don’t contain any NRPS genes! As far as is known, this makes these fungi unique. They seem to translate proproteins directly from mRNA. In other words, amatoxins and phallotoxins are likely to be directly encoded in the genome whereas other fungi encode NRPSs. This is an innovation not known from any other organism in kingdom Fungi.

11. POP goes the toxin… As will be explained in the paper, the initial peptide is probably cleaved by a prolyl oligopeptidase. Genes encoding POPs are found in other basidiomycetes (but few other fungi) as well as animals. POPs are enzymes that can only cleave peptides fewer than 40 amino acids in length. The particular POP involved here cleaves the peptide at a proline. The authors have not yet identified a particular POP involved in this pathway.

12. An Update from the PI …This is speculative, but I have a hard time believing that Amanita section Phalloideae came up, de novo, with this pathway that exists in no other fungus - if for no other reason than that Galerina, Lepiota and Conocybe species produce amatoxins. We are in the process of sequencing Galerina, and are hoping to have its amatoxin gene by the end of the year. What I am guessing will be the case is that we'll have something recognizably similar to Amanita's - maybe MSDLQ (e.g.) instead of MSDIN, which would be chemically similar but would suffice to render our Amanita-derived PCR primers and possibly Southerns of no use on Galerina. Once we have Galerina, IF things are as I expect, we would then be able to formulate rules to search the extant basidiomycete genomes - such as "ORF 100-150 bp, beginning with Mxxxx, containing at least two prolines, separated by x-y residues". Since Amanita's "MSDIN" is clearly not the whole story, I am hoping Galerina will alllow us to narrow and focus the search parameters a little. I guess all this is just a really verbose and meandering way of saying, I think there's a lot going on that we're not aware of in the basidiomycota, and I wouldn't be surprised if a propeptide-based mechanism of generating peptide secondary metabolites is widespread, possibly replacing ascomycetes' NRPSs. — Personal correspondence from Heather Hallen, 11/29/07

13. Fig. 1

14. Fig. 2

15. Fig. 3

16. Fig. 4

17. Fig. 5

18. Fig. 6

19. Supporting Information http://www.pnas.org/cgi/data/0707340104/DC1/1