Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode ␣-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. ␣-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable ''toxin'' region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.amanitin ͉ cyclic peptide ͉ phalloidin ͉ phallotoxin ͉ amatoxin

High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.

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The cost of enzymes for converting plant biomass materials to fermentable sugars is a major impediment to the development of a practical lignocellulosic ethanol industry. Research on enzyme optimization with the goal of reducing the cost of converting biomass materials such as corn stover into glucose, xylose, and other sugars is being actively pursued in private industry, academia, and government laboratories. Under the auspices of the Department of Energy Great Lakes Bioenergy Research Center, we are taking several approaches to address this problem, including "bioprospecting" for superior key enzymes, protein engineering, and high-level expression in plants. A particular focus is the development of synthetic enzyme mixtures, in order to learn which of the hundreds of known enzymes are important and in what ratios. A core set comprises cellobiohydrolase, endoglucanase, β-glucosidase, endoxylanase, and β-glucosidase. Accessory enzymes include esterases, proteases, nonhydrolytic proteins, and glycosyl hydrolases that cleave the less frequent chemical linkages found in plant cell walls.