Background: Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway. Results: The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes α-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the housekeeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome. Conclusions: To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantlyrelated families.
Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms.
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