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Genes and evolutionary fates of the amanitin biosynthesis pathway in poisonous mushrooms - PubMed

  • ️Sat Jan 01 2022

Genes and evolutionary fates of the amanitin biosynthesis pathway in poisonous mushrooms

Hong Luo et al. Proc Natl Acad Sci U S A. 2022.

Abstract

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

Keywords: Amanita; Galerina; Lepiota; gene cluster; genome.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

The amanitin biosynthesis locus and biosynthetic genes in Galerina species. (A) Amanitin biosynthetic genes are color-coded (red, green, blue, and black). Candidate biosynthetic genes are marked as light blue arrowheads. The amanitin biosynthetic loci were aligned for three Galerina genomes. Gene number suffix: position upstream (-) of GmAMA1-1; the two named downstream genes are not numbered based on position. Lines connect the genes (dots) with the highest homology within the genomes. (B) Deletion of GmFMO1 in G. marginata results in termination of α-amanitin production. In liquid chromatography–mass spectrometry (LC-MS) analysis: solid line denotes OD295 for wild type; red dashed line denotes OD295 for GmFMO1 mutant. In the DNA blot analysis, the wild type and the mutant are shown, with hph for the hygromycin B marker. (C) Deletion of GmP450-29 in G. marginata results in termination of α-amanitin production. In the LC-MS analysis: solid line denotes OD295 for wild type; red dashed line denotes OD295 for GmP450-29 mutant. In the DNA blot analysis, the wild type and the mutant are shown, with hph for the hygromycin B marker.

Fig. 2.
Fig. 2.

The amanitin biosynthesis loci. Contigs or scaffolds are indicated as blocks with combined sizes on the right (percentage of genome assemblies in parentheses); the display is in scale, with details in a magnified view for the locus in Galerina species. (A) Distribution of amanitin biosynthesis genes in the genome of lethal Amanita species. (B) Distribution of biosynthesis genes in lethal Lepiota species. (C) Distribution of the biosynthesis genes in lethal Galerina species. Homologs of MSDIN, POPB, P450-29, and FMO1 marked in red, green, blue, and black, respectively.

Fig. 3.
Fig. 3.

MSDIN core peptide sequences and known cyclic peptides in lethal Amanita, Galerina, and Lepiota. Core peptides (the amino acid residues in the cyclic peptide products) of known MSDINs across the three genera are listed on top of each column (shaded in gray). The α-amanitin–forming peptide is marked in red, which is the only one shared across the three genera. Shared core peptides across species are indicated with dots of the same colors in front of the peptides.

Fig. 4.
Fig. 4.

Southern blotting of amanitin biosynthesis CYP450 genes (AbP450-1, AbP450-2, and AbP450-3) in Amanita, Lepiota, and Galerina species. (A) Map of the genomic lambda clone PA1 from A. bisporigera. DNA blots show taxonomic distribution of AbP450-1 (B), AbP450-2 (C), and AbP450-3 (D). Lanes: 1, A. bisporigera; 2, A. phalloides; 3, A. porphyria; 4, A. franchetii; 5, G. marginata; 6, L. brunneoincarnata. Amanitin-producing species are marked with asterisks. PHA1-1 and PHA1-2 are the two copies of the phallacidin-encoding MSDIN gene.

Fig. 5.
Fig. 5.

Evolutionary outcome of the amanitin biosynthesis pathway in mushrooms. The modes of nutrition, mycorrhizal symbiosis, wood decomposition, and soil saprotroph are indicated below the genomes. Within the representative genomes for Amanita, Galerina, and Lepiota, predicted genes are indicated as blue lines, GC content as red lines, and GC skew as black lines. Four toxin biosynthetic genes are color-labeled to show their respective arrangements in the genomes. Genetic distances of the biosynthetic genes are similarly color-coded as lines connecting genome circles (housekeeping rpb2 indicated as black lines). The thick red arrows represent the best hypothesis from comprehensive evaluations.

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