Wang-Qiu Deng

Illumina-based de novo transcriptome sequencing and analysis of Amanita exitialis basidiocarps.

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.

Peptide toxin components of Amanita exitialis basidiocarps

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, β-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.

The molecular diversity of toxin gene families in lethal Amanita mushrooms.

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.

A new species of Amanita section Lepidella from South China

Amanita macrocarpa from Guangdong Province of China is described. The new species is a large, brownish orange to light brown mushroom, characterized by numerous pyramidal warts on the pileus, yellowish lamellae, a large annulus at the middle of the stipe, and amyloid ellipsoidal basidiospores. Phylogenetic analyses of the new species and related taxa based on the large subunit of nuclear ribosomal DNA (LSU) and the internal transcribed spacer (ITS) sequences are provided. Morphological and molecular data show that A. macrocarpa is a member of Amanita, section Lepidella, and clearly different from any known taxon of the section.

Amanita cinereovelata , a new species of Amanita section Lepidella from Bangladesh

Amanita cinereovelata is described and illustrated as a new species of Amanita sect. Lepidella from Bangladesh. The species is characterized by its small to medium-sized basidiomata, gray to lilac-gray pulverulent-floccose volval remnants on the pileus, a bulbous stipe base, globose to subglobose amyloid basidiospores, the presence of clamps at the bases of basidia, and an association with Shorea robusta. Molecular phylogenetic analysis indicates that A. cinereovelata is a species sister to A. eriophora, A. longipes, A. tephrea and A. vestita. Comparisons with morphologically similar taxa are provided.

Molecular cloning of α-amanitin and characterization of its expression pattern in different parts and development stages of Amanita exitialis fruitbody

Amanita exitialis is a lethal mushroom in East Asia. α-Amanitin is one kind of the leading peptide toxins responsible for mushroom poisoning, which are mainly from the fatal amanitae. In the study, the full-length cDNA sequences of α-amanitin (α-AMA) and phallacidin (PHA) from A. exitialis were cloned and analyzed; in addition, the expression patterns of α-AMA in three parts as well as four developmental stages of the fruitbody were first characterized by quantitative real-time PCR. Results demonstrate that α-AMA and PHA were highly homologous to the toxin genes reported from A. bisporigera; the α-AMA could be expressed in all parts and stages of the fruitbody, from late elongation stage to late mature stage, α-AMA showed the highest expression level in the pileus, followed by the stipe and volva in sequence, while at the early elongation stage, the highest expression level was found in the stipe, followed by the pileus and volva. Further analysis revealed that the higher expression level of α-AMA was closely associated with the development stages, and the expression of α-AMA was accumulated in the more vigorous growth parts and stages, from which could be inferred that α-AMA might play an important role in the development of A. exitialis.

Marasmius albopurpureus , a new species of section Globulares from Baili Island, China

Marasmius albopurpureus, a new species belonging to section Globulares, was found on a subtropical island, Baili Island, in southern China. It is characterized by its white to purple rugulose to sulcate pileus, distant and sometimes underdeveloped lamellae, and long basidiospores. It is described, illustrated and compared with similar taxa according to the morphological and molecular data.

Molecular cloning and the expression pattern of AePOPB involved in the α-amanitin biosynthesis in Amanita exitialis fruiting bodies

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.

Whole Genome Sequence of an Edible and Potential Medicinal Fungus, Cordyceps guangdongensis

Cordyceps guangdongensis is an edible fungus which was approved as a novel food by the Chinese Ministry of Public Health in 2013. It also has a broad prospect of application in pharmaceutical industries, with many medicinal activities. In this study, the whole genome of C. guangdongensis GD15, a single spore isolate from a wild strain, was sequenced and assembled with Illumina and PacBio sequencing technology. The generated genome is 29.05 Mb in size, comprising nine scaffolds with an average GC content of 57.01%. It is predicted to contain a total of 9150 protein-coding genes. Sequence identification and comparative analysis indicated that the assembled scaffolds contained two complete chromosomes and four single-end chromosomes, showing a high level assembly. Gene annotation revealed a diversity of transposons that could contribute to the genome size and evolution. Besides, approximately 15.57% and 12.01% genes involved in metabolic processes were annotated by KEGG and COG respectively. Genes belonging to CAZymes accounted for 3.15% of the total genes. In addition, 435 transcription factors, involved in various biological processes, were identified. Among the identified transcription factors, the fungal transcription regulatory proteins (18.39%) and fungal-specific transcription factors (19.77%) represented the two largest classes of transcription factors. This genomic resource provided a new insight into better understanding the relevance of phenotypic characters and genetic mechanisms in C. guangdongensis.

Genetic Diversity Analysis of Hypsizygus marmoreus with Target Region Amplification Polymorphism

Hypsizygus marmoreus is an industrialized edible mushroom. In the present paper, the genetic diversity among 20 strains collected from different places of China was evaluated by target region amplification polymorphism (TRAP) analysis; the common fragment of TRAPs was sequenced and analyzed. Six fixed primers were designed based on the analysis of H. marmoreus sequences from GenBank database. The genomic DNA extracted from H. marmoreus was amplified with 28 TRAP primer combinations, which generated 287 bands. The average of amplified bands per primer was 10.27 (mean polymorphism is 69.73%). The polymorphism information content (PIC) value for TRAPs ranged from 0.32 to 0.50 (mean PIC value per TRAP primer combination is 0.48), which indicated a medium level of polymorphism among the strains. A total of 36 sequences were obtained from TRAP amplification. Half of these sequences could encode the known or unknown proteins. According to the phylogenetic analysis based on TRAP result, the 20 strains of H. marmoreus were classified into two main groups.