Differential Expression of Amanitin Biosynthetic Genes and Novel Cyclic Peptides in Amanita molliuscula

Abstract

Amanita molliuscula is a basal species of lethal Amanita and intrigues the field because it does not produce discernable α-amanitin when inspected by High Performance Liquid Chromatography (HPLC), which sets it apart from all known amanitin-producing (lethal) Amanita species. In order to study the underlining genetic basis of the phenotype, we sequenced this species through PacBio and Illumina RNA-Seq platforms. In total, 17 genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides) were found in the genome and 11 of them were expressed at the transcription level. The expression pattern was not even but in a differential fashion: two of the MSDINs were highly expressed (FPKM value > 100), while the majority were expressed at low levels (FPKM value < 1). Prolyl oligopeptidease B (POPB) is the key enzyme in the amanitin biosynthetic pathway, and high expression of this enzyme was also discovered (FPKM value > 100). The two MSDINs with highest transcription further translated into two novel cyclic peptides, the structure of which is distinctive from all known cyclic peptides. The result illustrates the correlation between the expression and the final peptide products. In contrast to previous HPLC result, the genome of A. molliuscula harbors α-amanitin genes (three copies), but the product was in trace amount indicated by MS. Overall, transcription of MSDINs encoding major toxins (α-amanitin, β-amanitin, phallacidin and phalloidin) were low, showing that these toxins were not actively synthesized at the stage. Collectively, our results indicate that the amanitin biosynthetic pathway is highly active at the mature fruiting body stage in A. molliuscula, and due to the differential expression of MSDIN genes, the pathway produces only a few cyclic peptides at the time.

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

“…Copies of identical MSDIN genes are often linked, which partly causes the patchy patterns. Recent studies showed that most of the MSDIN s are expressed at the transcription level ( 16 , 20 ), and through a genome-guided approach we found 14 cyclic peptides in 5 Amanita species ( 20 , 21 ). Known cyclic peptides are shown in Fig.…”

“…Whole Genome Shotgun assemblies have been deposited at Data Bank of Japan (DDBJ)/European Nucleotide Archive (ENA)/GenBank under the accession number JAEBUT000000000. We combined these genomes with the genome of G. marginata CBS 339.88, available from the Joint Genome Institute (JGI) MycoCosm database ( 19 ), and the Amanita and Lepiota genomes previously published ( 9 , 11 , 16 , 20 , 21 ). The completeness of the gene repertoires of 13 sequenced genomes, based on Benchmarking Universal Single-Copy Orthologs (BUSCO) and CEGMA (Core Eukaryotic Genes Mapping Approach) analyses, are provided in SI Appendix , Table S1 .…”

“…Besides the above genomes sequenced in this study, those of Amanita molliuscula , A. rimosa , A. pallidorosea , A. subjunquillea , Amanita exitialis , A. phalloides , A. bisporigera , and L. venenata were generated from previous studies ( 9 , 11 , 16 , 20 , 21 ). In addition, a genome of a monokaryotic strain of G. marginata (France, CBS 339.88) was acquired through the Joint Genome Institute ( 19 ).…”