The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes

Abstract

The biosynthetic pathway for amanitins and related cyclic peptides in deadly <i>Amanita</i> (<i>Amanitaceae</i>) mushrooms represents the first known ribosomal cyclic peptide pathway in the <i>Fungi</i>. Amanitins are found outside of the genus in distantly related agarics <i>Galerina</i> (<i>Strophariaceae</i>) and <i>Lepiota</i> (<i>Agaricaceae</i>). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, <i>A. pallidorosea</i> and <i>A. subjunquillea</i>, were deep sequenced, and sequences of biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl oligopeptidases (<i>POPA</i> and <i>POPB</i>) were obtained. The two <i>Amanita</i> species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from <i>L. brunneoincarnata</i> basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl oligopeptidase B (POPB), a key enzyme in the biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of <i>POPB</i> and <i>POPA</i> based on both coding and amino acid sequences showed very different results: while <i>POPA</i> genes clearly reflected the phylogeny of the host species, <i>POPB</i> did not; strikingly, it formed a well-supported monophyletic clade, despite that the species belong to different genera in disjunct families. <i>POPA</i>, a known house-keeping gene, was shown to be restricted in a branch containing only <i>Amanita</i> species and the phylogeny resembled that of those <i>Amanita</i> species. Phylogenetic analyses of MSDIN and <i>POPB</i> genes showed tight coordination and disjunct distribution. A <i>POPB</i> gene tree was compared with a corresponding species tree, and distances and substitution rates were compared. The result suggested <i>POPB</i> genes have significant smaller distances and rates than the house-keeping <i>rpb2</i>, discounting massive gene loss. Under this assumption, the incongruency between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster <i>Galerina</i> and <i>Amanita POPB</i> genes, while <i>Lepiota POPB</i> is distinct. Our result suggests that horizontal gene transfer (HGT), at least between <i>Amanita</i> and <i>Galerina,</i> was involved in the acquisition of <i>POPB</i> genes, which may shed light on the evolution of the α-amanitin biosynthetic pathway.