Novel cyclic peptides from lethal Amanita species through a genomic approach, and major peptide toxins in the ectomycorrhizal association

Abstract

Background: Most species in the genus Amanita are ectomycorrhizal fungi, and the cyclic peptide toxins that some species produce are notoriously deadly. In total, around 25 of these peptides were found in the fruiting bodies over the past 82 years, and whether any of them are present in the mycorrhizae is unknown. Reportedly, sequenced lethal Amanita genomes harbor a significant number of precursor genes of MSDIN family, indicating there could be a much larger capacity for cyclic peptide production in these mushrooms. However, it is largely unknown that to what extent these genes are transcribed, and further, translated into true cyclic peptides. Method:In this study, three poisonous Amanita species, A. rimosa, A. exitialis and A. subjunquillea, were sequenced through PacBio and Illumina techniques. For expression analysis, one strain of A.subjunquillea was sequenced through RNA-Seq. A genome-guided approach was adopted to identify cyclic peptides by coupling predicted toxin-biosynthetic genes with mass spectrometry (MS and MS/MS). To investigate whether any of the toxins were express in the microbiome, profiling of known major toxins was conducted on A. subjunquillea mycorrhizae via HRMS and gene cloning. Results:The resultant genomes showed significant potential to produce known and unknown cyclic peptides. Together with our 2 previously sequenced genomes, in total 37 unknown MSDIN genes were discovered. Expression of over 90% of the MSDIN genes was demonstrated in two strains of A. subjunquillea. Through the genome-guided approach, 12 MSDIN genes were found to produce true, novel cyclic peptides with no additional posttranslational modifications. When the ectomycorrhizae of A. subjunquillea were analyzed by MS, all major toxins were detected. The corresponding MSDINs for these cyclic peptides were successfully cloned directly from the mycorrhizae. Conclusions:The genome-guided approach provided a speedy method to identify cyclic peptides both in Amanita mushrooms and in the ectomycorrhizae. In this study, a significant number of novel MSDIN genes were discovered, most of which were found to be expressed in the tested species. The identification of the 12 novel cyclic peptides strongly suggests that Amanita species possess a much larger reservoir of these peptides than previously thought. This is the first report to demonstrate that the cyclic peptides in Amanita species are expressed in the mycorrhizal association. All four major