Yong-Jie Zhang

Ophiocordyceps sinensis, the flagship fungus of China: terminology, life strategy and ecology

The Chinese caterpillar fungus, Ophiocordyceps sinensis (syn. Cordyceps sinensis), is one of the most famous and perhaps the most expensive fungal species in the world. Its biology largely remains a secret, and its commercial cultivation is still a dream. Owing to its medicinal, economic, social and ecological importance, and its limited distribution mainly in China, O. sinensis is herein nominated as the national fungus of China and the fungus of the year (2012) for the journal Mycology. To clarify the confusion, a standard nomenclature is proposed and recommended in this paper for a comprehensive understanding of the terms related to Cordyceps sensu lato and O. sinensis. We also review recent research on the life cycle and distribution of this fungus, parasitism of ghost moths by the fungus, the microbial community of natural Chinese cordyceps and its insect hosts, as well as the secondary metabolites produced by the fungi isolated from natural Chinese cordyceps. By taking advantage of various expertises as well as the government support, we believe that the biological secrets of O. sinensis will be unravelled gradually, and the sustainable development and utilization of this traditional medicine will be fully achieved in the future.

Comparison of mitochondrial genomes provides insights into intron dynamics and evolution in the caterpillar fungus Cordyceps militaris

Intra-specific comparison of mitochondrial genomes can help elucidate the evolution of a species, however it has not been performed for hypocrealean fungi that form diverse symbiotic associations with other organisms. In this study, comparative analyses of three completely sequenced mitochondrial genomes of a hypocrealean fungus, Cordyceps militaris, the type species of Cordyceps genus, revealed that the introns were the main contributors to mitochondrial genome size variations among strains. Mitochondrial genes in C. militaris have been invaded by group I introns in at least eight positions. PCR assays of various C. militaris isolates showed abundant variations of intron presence/absence among strains at seven of the eight intronic loci. Although the ancestral intron pattern was inferred to contain all eight introns, loss and/or gain events occurred for seven of the eight introns. These introns invaded the C. militaris mitochondrial genome probably by horizontal transfer from other fungi, and intron insertions into intronless genes in C. militaris were accompanied by co-conversions of upstream exon sequences especially for those introns targeting protein-coding genes. We also detected phylogenetic congruence between the intron and exon trees at each individual locus, consistent with the ancestral mitochondria of C. militaris as having all eight introns. This study helps to explain the evolution of C. militaris mitochondrial genomes and will facilitate population genetic studies of this medicinally important fungus.

Purification and characterization of a novel immunomodulatory protein from the medicinal mushroom Trametes versicolor

Bioactive proteins represent an important group of functional agents in medicinal mushrooms. Trametes versicolor (L.) Lloyd is a mushroom frequently used in traditional Chinese medicine for its anti-tumor and immunomodulatory activities. A new immunomodulatory protein from T. versicolor, named TVC, was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed a single band with a molecular weight of 15.0 kD. Native polyacrylamide gel analysis revealed a band at 30 kD, indicating that TVC exists in solution as a homodimer. Isoelectric focusing showed that TVC was an acidic protein with an isoelectric point of 4.0. TVC was found to lack carbohydrate modifications (based on periodic acid/Schiff staining) and it does not agglutinate mouse red blood cells, suggesting that TVC is not a lectin-like protein. Biological activity assays demonstrated that TVC can enhance the proliferation of splenocytes, while it has no stimulatory effects on CD4+ and CD8+ T cells. TVC markedly increases the proliferation of human peripheral blood lymphocytes in a dose-dependent manner and enhances the production of both nitric oxide and tumor necrosis factor-alpha by lipopolysaccharide-induced murine macrophages. The results indicate that TVC is an immunostimulant that can boost immune response. Comparison of the N-terminal amino acid residues and mass spectrometry results with the protein database revealed no homologous proteins.

Phylogeography and evolution of a fungal-insect association on the Tibetan Plateau

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high‐frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis–ghost moth parasitoidism on the Tibetan Plateau.

Cloning and analysis of the MAT1-2-1 gene from the traditional Chinese medicinal fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.

Complete mitochondrial genome of the endophytic fungus Pestalotiopsis fici: features and evolution

Endophytic fungi (EF) live within plants and have profound impacts on plant communities. They are astonishingly diverse but poorly studied at the genome level. Herein, we assembled the mitochondrial genome (mitogenome) of the EF Pestalotiopsis fici, annotated and compared it with those of other relatives to better understand the evolution of the EF lineage. Except for standard fungal mitochondrial genes, the 69,529-bp circular mitogenome of P. fici harbors 18 introns acquired possibly through lateral transfer from other fungi and nine free-standing open reading frames with some scarcely seen in fungal mitogenomes. BLAST analysis detected no obvious duplication events of large fragments between mitochondrial and nuclear genomes of the fungus. Transcription analyses validated the expression of all mitochondrial genes, while most genes showed higher expression on rice than in two other media. The mitogenome of P. fici is highly syntenic with the Xylariales species Annulohypoxylon stygium and the endophyte Epichloe festucae var. lolii, but lacks synteny with another endophyte Penicillium polonicum. This study reports the first mitogenome of Pestalotiopsis and the third published mitogenome from an EF and provides insights into the evolution of the EF lineage.

The complete mitochondrial genome of the nematode endoparasitic fungus Hirsutella minnesotensis

Abstract In this study, the complete mitochondrial genome of a nematode fungal pathogen, Hirsutella minnesotensis, is sequenced. The circular mitogenome of H. minnesotensis is 52,245 bp in length and consists of 2 rRNA, 25 tRNA and 14 standard protein-coding genes of the oxidative phosphorylation system as well as four additional free-standing ORFs encoding for an endonuclease or a hypothetical protein. Seven genes (rnl, cob, cox1, nad3, nad4, nad4L and nad5) are invaded by group I or unclassified introns, and these introns carry ORFs of ribosomal protein S3 and GIY-YIG/LAGLIDADG endonucleases or hypothetical proteins. Evidence of intron degeneration is significant for the nad4L intron and cox1-i1 due to unexpected stop codons and/or frame shifting. Phylogenetic analysis based on concatenated protein sequences confirms H. minnesotensis as a member of the fungal order Hypocreales. In this study, we present the complete mitogenome sequence of H. minnesotensis, which is the first complete mitogenome of the family Ophiocordycipitaceae.

The mitochondrial genome of the nematode endoparasitic fungus Hirsutella rhossiliensis

Abstract In this study, we report the complete mitochondrial genome of Hirsutella rhossiliensis (Ophiocordycipitaceae, Hypocreales, Ascomycota). We construct the mitochondrial DNA genome organization of 62 483 bp in length of H. rhossiliensis by using the whole-genome resequencing method. Conserved genes including the large and small rRNA subunits, 26 tRNA and 14 protein-coding genes are identified. These protein-coding genes utilize ATG, GTG or TTG as initiation codons and TAA or TAG as termination codons. Moreover, we detect 10 group I introns and one unclassified intron in six genes (rnl, cob, cox1, cox3, nad1 and nad5) encoding ORFs of ribosomal protein S3 and GIY-YIG/LAGLIDADG endonucleases or hypothetical proteins. This mitochondrial genome will be useful in understanding the distribution and genetic diversity of this species.

Population Genetics of Hirsutella rhossiliensis, a Dominant Parasite of Cyst Nematode Juveniles on a Continental Scale

ABSTRACT Hirsutella rhossiliensis is a parasite of juvenile nematodes, effective against a diversity of plant-parasitic nematodes. Its global distribution on various nematode hosts and its genetic variation for several geographic regions have been reported, while the global population genetic structure and factors underlying patterns of genetic variation of H. rhossiliensis are unclear. In this study, 87 H. rhossiliensis strains from five nematode species (Globodera sp., Criconemella xenoplax, Rotylenchus robustus, Heterodera schachtii, and Heterodera glycines) in Europe, the United States, and China were investigated by multilocus sequence analyses. A total of 280 variable sites (frequency, 0.6%) at eight loci and six clustering in high accordance with geographic populations or host nematode-associated populations were identified. Although H. rhossiliensis is currently recognized as an asexual fungus, recombination events were frequently detected. In addition, significant genetic isolation by geography and nematode hosts was revealed. Overall, our analyses showed that recombination, geographic isolation, and nematode host adaptation have played significant roles in the evolutionary history of H. rhossiliensis. IMPORTANCE H. rhossiliensis has great potential for use as a biocontrol agent to control nematodes in a sustainable manner as an endoparasitic fungus. Therefore, this study has important implications for the use of H. rhossiliensis as a biocontrol agent and provides interesting insights into the biology of this species.

Fungal mitochondrial genomes and genetic polymorphisms

Mitochondria are the powerhouses of eukaryotic cells, responsible for ATP generation and playing a role in a diversity of cellular and organismal functions. Different from the majority of other intracellular membrane structures, mitochondria contain their own genetic materials that are capable of independent replication and inheritance. In this mini-review, we provide brief summaries of fungal mitochondrial genome structure, size, gene content, inheritance, and genetic variation. We pay special attention to the relative genetic polymorphisms of the mitochondrial vs nuclear genomes at the population level within individual fungal species. Among the 20 species/groups of species reviewed here, there is a range of variation among genes and species in the relative nuclear and mitochondrial genetic polymorphisms. Interestingly, most (15/20) showed a greater genetic diversity for nuclear genes and genomes than for mitochondrial genes and genomes, with the remaining five showing similar or slower nuclear genome genetic variations. This fungal pattern is different from the dominant pattern in animals, but more similar to that in plants. At present, the mechanisms for the variations among fungal species and the overall low level of mitochondrial sequence polymorphisms are not known. The increasing availability of population genomic data should help us reveal the potential genetic and ecological factors responsible for the observed variations.