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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.

Some but not all of the species of 'little brown mushrooms' in the genus Galerina contain deadly amatoxins at concentrations equaling those in the death cap, Amanita phalloides. However, Galerina's ~300 species are notoriously difficult to identify by morphology, and the identity of toxin-containing specimens has not been verified with DNA barcode sequencing. This left open the question of which Galerina species contain toxins and which do not. We selected specimens for toxin analysis using a preliminary phylogeny of the fungal DNA barcode region, the ribosomal internal transcribed spacer (ITS) region. Using liquid chromatography/mass spectrometry, we analyzed amatoxins from 70 samples of Galerina and close relatives, collected in western British Columbia, Canada. To put the presence of toxins into a phylogenetic context, we included the 70 samples in maximum likelihood analyses of 438 taxa, using ITS, RNA polymerase II second largest subunit gene (RPB2), and nuclear large subunit ribosomal RNA (LSU) gene sequences. We sequenced barcode DNA from types where possible to aid with applications of names. We detected amatoxins only in the 24 samples of the G. marginata s.l. complex in the Naucoriopsis clade. We delimited 56 putative Galerina species using Automatic Barcode Gap Detection software. Phylogenetic analysis showed moderate to strong support for Galerina infrageneric clades Naucoriopsis, Galerina, Tubariopsis, and Sideroides. Mycenopsis appeared paraphyletic and included Gymnopilus. Amatoxins were not detected in 46 samples from Galerina clades outside of Naucoriopsis or from outgroups. Our data show significant quantities of toxin in all mushrooms tested from the G. marginata s.l. complex. DNA barcoding revealed consistent accuracy in morphology-based identification of specimens to G. marginata s.l. complex. Prompt and careful morphological identification of ingested G. marginata s.l. has the potential to improve patient outcomes by leading to fast and appropriate treatment., Competing Interests: The authors have declared that no competing interests exist.

Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita β-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas., (Copyright © 2019. Published by Elsevier Ltd.)

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis., (Copyright © 2018. Published by Elsevier B.V.)

Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

Mushrooms in lethal Amanita species are responsible for a large number of food poisoning cases and deaths. However, the diversity of the toxins in these mushrooms remains largely unknown. This study analyzed the gene families of toxins found in 6 lethal Amanitae from Asia and Europe. Fifty-four gene sequences were obtained, accounting for 70.1% of the known MSDIN family members. Of the 54 gene sequences, 20 encode α-amanitin, 5 encode β-amanitin, 16 encode phallacidin, and 13 encode peptides of unknown functions. Bayesian analysis of MSDIN family members identified differences in the number of toxin genes in different toxin families among the Amanita species. Ten of the 13 peptides of unknown functions were closely related to known phallotoxins, while the remaining 3 were similar to amatoxins. The α-AMA tree indicated that there were significant differences between the Amanita and Galerina species. However, both the α-AMA and PHA trees showed that these toxin genes have similar upstream and downstream motif sequences among the Amanita species. This study greatly enriches the available diversity information regarding toxin gene families in lethal Amanita mushrooms, and could lay a strong foundation for further research about the evolution of Amanita toxin genes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Amanita exitialis is a lethal mushroom that was first discovered in Guangdong Province, China. The high content of amanitin in its basidiocarps makes it lethal to humans. To comprehensively characterize the A. exitialis transcriptome and analyze the Amanita toxins as well as their related gene family, transcriptome sequencing of A. exitialis was performed using Illumina HiSeq 2000 technology. A total of 25,563,688 clean reads were collected and assembled into 62,137 cDNA contigs with an average length of 481 bp and N50 length of 788 bp. A total of 27,826 proteins and 39,661 unigenes were identified among the assembled contigs. All of the unigenes were classified into 166 functional categories for understanding the gene functions and regulation pathways. The genes contributing to toxic peptide biosynthesis were analyzed. From this set, eleven gene sequences encoding the toxins or related cyclic peptides were discovered in the transcriptome. Three of these sequences matched the peptide toxins α-amanitin, β-amanitin, and phallacidin, while others matched amanexitide and seven matched unknown peptides. All of the genes encoding peptide toxins were confirmed by polymerase chain reaction (PCR) in A. exitialis, and the phylogenetic relationships among these proprotein sequences were discussed. The gene polymorphism and degeneracy of the toxin encoding sequences were found and analyzed. This study provides the first primary transcriptome of A. exitialis, which provided comprehensive gene expression information on the lethal amanitas at the transcriptional level, and could lay a strong foundation for functional genomics studies in those fungi., (© 2013.)

We present a microfluidic electroporation device with a comb electrode layout fabricated in polydimethylsiloxane (PMDS) and glass. Characterization experiments with HeLa cells and fluorescent dextran show efficient delivery (∼95%) with low toxicity (cell viability ∼85%) as well as rapid pore closure after electroporation. The activity of delivered molecules is also verified by silencing RNA (siRNA) studies that demonstrate gene knockdown in GFP expressing cells. This simple, scalable approach to microfluidic, flow-through electroporation could facilitate the integration of electroporation modules within cell analysis devices that perform multiple operations.

Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as alpha- and beta-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys crossbridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34-35 amino acids in length and have no predicted signal peptides. The genes for alpha-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable "toxin" region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide.

Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode alpha-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. alpha-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable "toxin" region capable of encoding a wide variety of peptides of 7-10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.

The effects of the RNA polymerase II (RNAPII) translocation inhibitors alpha amanitin and 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) and an siRNA targeting p300 on the presence of RNAPII, p300, hyperacetylated H4 and H3 and unmodified H4 and H3 in transcribing simian virus 40 (SV40) minichromosomes were determined. Following treatment with alpha amanitin we observed a profound reduction in the occupancy of the promoter by RNAPII, the loss of p300 from chromatin fragments containing RNAPII, and an increase in the amount of unmodified H4 and H3 associated with the RNAPII. Treatment with DRB had little effect on the presence of RNAPII or p300 but also resulted in a significant increase in the amount of unmodified H4 and H3 present in chromatin fragments associated with RNAPII. Following treatment with a p300 small interfering RNA (siRNA), we observed a significant decrease in late transcription and a corresponding reduction in the amounts of p300 and hyperacetylated histones associated with the transcribing SV40 minichromosomes. We conclude that in transcribing SV40 minichromosomes histone hyperacetylation and deacetylation is dependent upon the presence of p300 and an as yet unknown histone deacetylase associated with the RNAPII complex that occurs coordinately as the RNAPII complex moves through a nucleosome.

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).

In eukaryotes, RNA polymerase (pol) II transcribes the protein-coding genes, whereas RNA pol I transcribes the genes that encode the three RNA species of the ribosome [the ribosomal RNAs (rRNAs)] at the nucleolus. Protozoan parasites of the order Kinetoplastida may represent an exception, because pol I can mediate the expression of exogenously introduced protein-coding genes in these single-cell organisms. A unique molecular mechanism, which leads to pre-mRNA maturation by trans-splicing, facilitates pol I-mediated protein-coding gene expression in trypanosomes. Trans-splicing adds a capped 39-nucleotide mini-exon, or spliced leader transcript, to the 5' end of the main coding exon posttranscriptionally. In other eukaryotes, the addition of a 5' cap, which is essential for mRNA function, occurs exclusively as a result of RNA pol II-mediated transcription. Given the assumption that cap addition represents the limiting factor, trans-splicing may have uncoupled the requirement for RNA pol II-mediated mRNA production. A comparison of the alpha-amanitin sensitivity of transcription in naturally occurring trypanosome protein-coding genes reveals that a unique subset of protein-coding genes-the variant surface glycoprotein (VSG) expression sites and the procyclin or the procyclic acidic repetitive protein (PARP) genes-are transcribed by an RNA polymerase that is resistant to the mushroom toxin alpha-amanitin, a characteristic of transcription by RNA pol I. Promoter analysis and a pharmacological characterization of the RNA polymerase that transcribes these genes have strengthened the proposal that the VSG expression sites and the PARP genes represent naturally occurring protein-coding genes that are transcribed by RNA pol I.

In Drosophila embryos the maternal/zygotic transition (MZT) in cell cycle control normally follows mitosis 13. Here we show that this transition requires degradation of two maternal mRNAs, string and twine, which encode Cdc25 phosphatases. Although twine is essential for meiosis and string is essential for most mitotic cycles, the two genes have mutually complementing, overlapping functions in the female germ line and the early embryo. Deletion of both gene products from the female germ line arrests germ-line development. Reducing the maternal dose of both products can lower the number of early embryonic mitoses to 12, whereas increasing maternal Cdc25(twine) can increase the number of early mitoses to 14. Blocking the activation of zygotic transcription stabilizes maternal string and twine mRNAs and also allows an extra maternal mitosis, which is Cdc25 dependent. We propose that Drosophila's MZT comprises a chain reaction in which (1) proliferating nuclei deplete factors (probably mitotic cyclins) required for cell cycle progression; (2) this depletion causes the elongation of interphases and allows zygotic transcription; (3) new gene products accumulate that promote degradation of maternal mRNAs, including string and twine; and (4) consequent loss of Cdc25 phosphatase activity allows inhibitory phosphorylation of Cdc2 by Dwee1 kinase, effecting G2 arrest. Unlike timing or counting mechanisms, this mechanism can compensate for losses or additions of nuclei by altering the timing and number of the maternal cycles and thus will always generate the correct cell density at the MZT.

The alpha-amanitin domain or domain f of the largest subunit of RNA polymerases is one of the most conserved of these enzymes. We have found that the C-terminal part of domain f can be swapped between yeast RNA polymerase II and III. An extensive mutagenesis of domain f of C160, the largest subunit of RNA polymerase III, was carried out to better define its role and understand the mechanism through which C160 participates in transcription. One mutant enzyme, C160-270, showed much reduced transcription of a non-specific template at low DNA concentrations. Abortive synthesis of trinucleotides in a dinucleotide-primed reaction proceeded at roughly wild-type levels, indicating that the mutation did not affect the formation of the first phosphodiester bond, but rather the transition from abortive initiation to processive elongation. In specific transcription assays, on the SUP4 tRNA gene, pausing was extended but the rate of RNA elongation between pause sites was not affected. Finally, the rate of cleavage of nascent RNA transcripts by halted mutant RNA polymerase was increased approximately 10-fold. We propose that the domain f mutation affects the transition between two transcriptional modes, one being adopted during abortive transcription and at pause sites, the other during elongation between pause sites.

Three wild strains of D. melanogaster have been identified which are resistant to normally lethal levels of the fungal toxin, alpha-amanitin. Dietary LD50S for Oregon-R, the reference strain, and Ama-KTT, Ama-MI and Ama-KLM, the resistant strains, are 1.2, 35, 30 and 10 micrograms alpha-amanitin/vial, respectively. Resistance in all three strains is a digenic trait, being determined by two independently acting dominant genes, Ama-1, Ama-2, either of which is sufficient to confer resistance and which are located at approximately 18.8 and 100.7, respectively, on chromosome 3. Resistance to alpha-amanitin in all three strains is apparently not mediated by modification of RNA polymerase form II nor by failure of toxin transport or by toxin inactivation.

Mutants of Caenorhabditis elegans resistant to alpha-amanitin have been isolated at a frequency of about 1.6 x 10(-6) after EMS mutagenesis of the wild-type strain, N2. Four new dominant resistance mutations have been studied genetically. Three are alleles of a previously identified gene, ama-1 IV, encoding the largest subunit of RNA polymerase II. The fourth mutation defines a new gene, ama-2 V. Unlike the ama-1 alleles, the ama-2 mutation exhibits a recessive-lethal phenotype. Growth and reproduction of N2 was inhibited at a concentration of 10 micrograms/ml amanitin, whereas ama-2/+ animals were inhibited at 100 micrograms/ml, and 800 micrograms/ml was required to inhibit growth of ama-1/+ larvae. We have also determined that two reference strains used for genetic mapping, dpy-11(e224)V and sma-1(e30)V, are at least four-fold more sensitive to amanitin that the wild-type strain. Using an amanitin-resistant ama-1(m118) or ama-1(m322) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. The frequency of EMS-induced lethal ama-1 mutations is approximately 1.7 x 10(-3), 1000-fold higher than the frequency of amanitin-resistance alleles. Nine of the lethal alleles are apparent null mutations, and they exhibit L1-lethal phenotypes at both 20 degrees and 25 degrees. Six alleles result in partial loss of RNA polymerase II function as determined by their sterile phenotypes at 20 degrees. All but one of these latter mutations exhibit a more severe phenotype at 25 degrees C. We have also selected seven EMS-induced revertants of three different ama-1 lethals. These revertants restore dominant resistance to amanitin. The selection for revertants also produced eight new dominant amanitin resistance alleles on the balancer chromosome, nT1.

The hypothesis of functional hemizygosity has been examined for the alpha-amanitin resistant (AmaR, a codominant marker) locus in a series of Chinese hamster cell lines. AmaR mutants were obtained from different cell lines, e.g., CHO, DHW, M3- 1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards alpha-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to alpha-amanitin inhibition, only about 50% of the activity is highly resistant in AmaR mutants of CHW and M3- 1 cell lines. The remaining activity in the latter cell lines shows alpha-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by alpha-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.

A fine-structure genetic map has been constructed for ama-1 IV, an essential gene in Caenorhabditis elegans encoding the amanitin-binding subunit of RNA polymerase II. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination frequencies with m118, a mutation that confers dominant resistance to alpha-amanitin. The 16 mutants, all isolated in the ama-1(m118) background, include 13 that are early larval lethals, and three that are mid-larval lethals, at 25 degrees. Six of the mutants exhibit temperature-dependence in the severity of their phenotype. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 X 10(-6). The segregation of the closely linked flanking markers, unc-17 and unc-5, revealed whether the lethal mutation was to the left or the right of m118. By adding the distances between the extreme left and right mutations, the ama-1 gene is estimated to be 0.011 map unit long, with m118 positioned 0.004 map unit from the left-most lethal mutation. To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication, mDp1[unc-17(e113) dpy-13(+) ama-1(+)]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the left-right order of the lethal mutations. The position of mutations within the gene is nonrandom. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.