Expansion and diversification of the MSDIN family of cyclic peptide genes in the poisonous agarics Amanita phalloides and A. bisporigera

Abstract

BackgroundThe cyclic peptide toxins of Amanita mushrooms, such as α-amanitin and phalloidin, are encoded by the “MSDIN” gene family and ribosomally biosynthesized. Based on partial genome sequence and PCR analysis, some members of the MSDIN family were previously identified in Amanita bisporigera, and several other members are known from other species of Amanita. However, the complete complement in any one species, and hence the genetic capacity for these fungi to make cyclic peptides, remains unknown.ResultsDraft genome sequences of two cyclic peptide-producing mushrooms, the “Death Cap” A. phalloides and the “Destroying Angel” A. bisporigera, were obtained. Each species has ~30 MSDIN genes, most of which are predicted to encode unknown cyclic peptides. Some MSDIN genes were duplicated in one or the other species, but only three were common to both species. A gene encoding cycloamanide B, a previously described nontoxic cyclic heptapeptide, was also present in A. phalloides, but genes for antamanide and cycloamanides A, C, and D were not. In A. bisporigera, RNA expression was observed for 20 of the MSDIN family members. Based on their predicted sequences, novel cyclic peptides were searched for by LC/MS/MS in extracts of A. phalloides. The presence of two cyclic peptides, named cycloamanides E and F with structures cyclo(SFFFPVP) and cyclo(IVGILGLP), was thereby demonstrated. Of the MSDIN genes reported earlier from another specimen of A. bisporigera, 9 of 14 were not found in the current genome assembly. Differences between previous and current results for the complement of MSDIN genes and cyclic peptides in the two fungi probably represents natural variation among geographically dispersed isolates of A. phalloides and among the members of the poorly defined A. bisporigera species complex. Both A. phalloides and A. bisporigera contain two prolyl oligopeptidase genes, one of which (POPB) is probably dedicated to cyclic peptide biosynthesis as it is in Galerina marginata.ConclusionThe MSDIN gene family has expanded and diverged rapidly in Amanita section Phalloideae. Together, A. bisporigera and A. phalloides are predicted to have the capacity to make more than 50 cyclic hexa-, hepta-, octa-, nona- and decapeptides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3378-7) contains supplementary material, which is available to authorized users.

Diversity of MSDIN family members in amanitin-producing mushrooms and the phylogeny of the MSDIN and prolyl oligopeptidase genes

“…It has been proven that lethal Amanita species are classified in the Amanita section Phalloideae and that these species contain the MSDIN gene family, allowing them to produce many small cyclopeptides, such as α-amanitin, on ribosomes [6,16]. In the present study, nine lethal Amanita species from China, including A. exitialis, A. fuliginea, A. mulliuscula, A. pallidorosea, A. rimosa, A. subfuliginea, A. subjunquillea, A. subpallidorosea and A. virosa, were proven to contain MSDIN genes, the same as other lethal Amanita species, such as A. bisporigera, described previously.…”

“…4 using degenerate primers [13]. Recently, Pulman et al (2016) showed by generating draft genome sequences of A. palloides and A. bisporigera that both possessed approximately 30 members of the MSDIN family, but only three of these genes were common to both [6]. Luo et al (2018) found 18 and 22 MSDIN genes in the A. subjunquillea and A. pallidorosea genomes through PacBio and Illumina sequencing, respectively [8].…”

“…Many new lethal Amanita and Lepiota species, including A. rimosa, A. subfuliginea, A. subpallidorosea, and L. venenata, etc., have been discovered over the past 10 years [17][18][19][20]. Meanwhile, in addition to the known twenty-two cyclopeptide toxins, some other new cyclopeptide substances, such as cycloamanide E and cycloamanide F in A. phalloides and amanexitide in A. exitialis, respectively, have been extracted and identified [6,21,22]. It has been reported that A. bisporigera and A. phalloides have large but essentially nonoverlapping potential for the biosynthesis of a variety of cyclopeptides, most of which are unknown according to predictions.…”

“…According to previous research, whole-genome sequencing has proven to be the most comprehensive and in-depth method to identify MSDIN genes or genes related to the cyclopeptide biosynthetic pathway in amanitin-producing mushrooms [6,8]. Nevertheless, compared with genome sequencing, transcriptome sequencing provides an alternative efficient and low-cost method to obtain functional gene data.…”

“…Nevertheless, compared with genome sequencing, transcriptome sequencing provides an alternative efficient and low-cost method to obtain functional gene data. To the best of knowledge, until now, only A. palloides and A. bisporigera from North America and A. exitialis from Asia have been studied using transcriptome sequencing [6,23].…”