A.S.M. Sonnenberg

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens

Agrobacterium tumefaciens is known to transfer parts of its tumor-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi. We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus. Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection marker is stable during mitosis and meiosis. The Agrobacterium system allows the transformation of both homokaryons and heterokaryons of A. bisporus. Also, both karyotypes of an heterokaryon can be transformed simultaneously. Furthermore, this is the first report on the transformation of vegetative mycelium of a commercial strain of A. bisporus.

Complementary symbiont contributions to plant decomposition in a fungus-farming termite

Significance Old World (sub)tropical fungus-growing termites owe their massive ecological footprints to an advanced symbiosis with Termitomyces fungi. They also have abundant gut bacteria, but the complementarity roles of these symbionts have remained unclear. We analyzed the genomic potential for biomass decomposition in a farming termite, its fungal symbiont, and its bacterial gut communities. We found that plant biomass conversion is mostly a multistage complementary cooperation between Termitomyces and gut bacteria, with termite farmers primarily providing the gut compartments, foraging, and nest building. A mature queen had highly reduced gut microbial diversity for decomposition enzymes, suggesting she had an exclusively fungal diet even though she may have been the source of the gut microbes of the colony’s first workers and soldiers. Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.

Fungal treated lignocellulosic biomass as ruminant feed ingredient: a review.

In ruminant nutrition, there is an increasing interest for ingredients that do not compete with human nutrition. Ruminants are specialists in digesting carbohydrates in plant cell walls; therefore lignocellulosic biomass has potential in ruminant nutrition. The presence of lignin in biomass, however, limits the effective utilization of cellulose and hemicellulose. Currently, most often chemical and/or physical treatments are used to degrade lignin. White rot fungi are selective lignin degraders and can be a potential alternative to current methods which involve potentially toxic chemicals and expensive equipment. This review provides an overview of research conducted to date on fungal pretreatment of lignocellulosic biomass for ruminant feeds. White rot fungi colonize lignocellulosic biomass, and during colonization produce enzymes, radicals and other small compounds to breakdown lignin. The mechanisms on how these fungi degrade lignin are not fully understood, but fungal strain, the origin of lignocellulose and culture conditions have a major effect on the process. Ceriporiopsis subvermispora and Pleurotus eryngii are the most effective fungi to improve the nutritional value of biomass for ruminant nutrition. However, conclusions on the effectiveness of fungal delignification are difficult to draw due to a lack of standardized culture conditions and information on fungal strains used. Methods of analysis between studies are not uniform for both chemical analysis and in vitro degradation measurements. In vivo studies are limited in number and mostly describing digestibility after mushroom production, when the fungus has degraded cellulose to derive energy for fruit body development. Optimization of fungal pretreatment is required to shorten the process of delignification and make it more selective for lignin. In this respect, future research should focus on optimization of culture conditions and gene expression to obtain a better understanding of the mechanisms involved and allow the development of superior fungal strains to degrade lignin in biomass.

Effects of mushroom‐derived β‐glucan‐rich polysaccharide extracts on nitric oxide production by bone marrow‐derived macrophages and nuclear factor‐κB transactivation in Caco‐2 reporter cells: Can effects be explained by structure?

Mushrooms are known for their immune-modulating and anti-tumour properties. The polysaccharide fraction, mainly beta-glucans, is responsible for the immune-modulating effects. Fungal beta-glucans have been shown to activate leukocytes, which depend on structural characteristics of beta-glucans. As edible mushrooms come in contact with the intestinal immune system, effects on enterocytes are also interesting. Our aim was to evaluate the effect of mushroom polysaccharide extracts varying in beta-glucan structure on nitric oxide production by bone marrow-derived macrophages (BMMs) from mice and on nuclear factor-kappaB transactivation in human intestinal Caco-2 cells. We demonstrated that extracts from Agaricus bisporus stimulated nitric oxide production by BMM, whereas extracts from Coprinus comatus and spores of Ganoderma lucidum had only minor effects. Furthermore, extracts of A. blazei Murill and Phellinus linteus had no effect at all. Almost all mushroom extracts lowered nuclear factor-kappaB transactivation in Caco-2 cells. Structural analysis of A. bisporus compared with A. blazei Murill suggests that branching of the beta-glucan chain is essential for immune-stimulating activity. In conclusion, extracts from A. bisporus activate BMM, without activating enterocytes. These characteristics make A. bisporus an attractive candidate as a nutritional compound to stimulate the immune response in depressed states of immunity.

AN EFFICIENT PROTOPLASTING REGENERATION SYSTEM FOR AGARICUS-BISPORUS AND AGARICUS-BITORQUIS

Conditions for efficient protoplasting and regeneration ofAgaricus bisporus andA. bitorquis are described. Especially forA. bisporus protoplasts, high regeneration frequencies were obtained (up to 30%). The protoplasting/regeneration system can be used for routine isolation of homokaryons ofA. bisporus. Such homokaryons, derived from protoplasts containing one type of nucleus only, can easily be identified by analyzing isoenzyme banding patterns.

Nucleotide sequence and expression of the gene encoding NADP(+) dependent glutamate dehydrogenase (gdhA) from Agaricus bisporus

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from anAgaricus bisporus recombinant phageλ library. The deduced amino acid sequence would specify a 457-amino acid protein that is highly homologous in sequence to those derived from previously isolated and characterized genes coding for microbial NADP+-GDH. The open reading frame is interrupted by six introns. None of the introns is located at either one of the positions of the two introns conserved in the corresponding open reading frames of the ascomycete fungiAspergillus nidulans andNeurospora crassa. Northern analysis suggests that theA. bisporus gdhA gene is transcriptionally regulated and that, unlike the case in ascomycetes, transcription of this gene is repressed upon the addition of ammonium to the culture.

Mitochondrial DNA variation in natural populations of the mushroom Agaricus bisporus

We investigated the patterns of mitochondrial DNA variation in the global population of the commercial mushroom Agaricus bisporus. Through the analysis of RFLPs among 441 isolates from nine countries in North America and Eurasia, we found a total of 140 mtDNA haplotypes. Based on population genetic analysis, there are four genetically distinct natural populations in this species, found in coastal California, desert California, France and Alberta (Canada). While 134 of the 140 mtDNA haplotypes were unique to single geographical regions, two mtDNA haplotypes, mt001 and mt002, were found in almost every population surveyed. These two mtDNA haplotypes also predominate among cultivars used throughout the world for at least the last two decades. These two mtDNA haplotypes are more similar to the cosmopolitan groups of mtDNA haplotypes than to the indigeneous clusters of mtDNA haplotypes from the two Californian regions.

Effect of fungal treatments of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and methane production

Maize stover, rice straw, oil palm fronds and sugarcane bagasse were treated with the white-rot fungi Ceriporiopsis subvermispora, Lentinula edodes, Pleurotus eryngii, or Pleurotus ostreatus at 24 °C for 0-6 weeks. The fungi increased total gas production from oil palm fronds by 68-132%, but none of the fungi improved the in vitro rumen fermentability of maize stover. C. subvermispora and L. edodes increased total gas production of sugarcane bagasse by 65-71%, but P. eryngii and P. ostreatus decreased it by 22-50%. There was a linear relationship (P<0.05) between the proportion of lignin in the original substrate and the increase in in vitro gas production observed for C. subvermispora and L. edodes treatments (R2=0.92 and 0.96, respectively). It is concluded that C. subvermispora and L. edodes have a particularly high potential to improve the nutritive value of highly lignified ruminant feeds.

Genome-wide survey of repetitive DNA elements in the button mushroom Agaricus bisporus

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of the genome-wide distribution of these elements and provide insights into potential mechanisms of genome plasticity. The present study uses in silico approaches to describe tandem repeats and transposable elements distribution in the genome of the button mushroom, Agaricus bisporus. Transposable elements comprised 12.43% of the assembled genome, and 66% of them were found clustered in the centromeric or telomeric regions. Methylation of retrotransposon has been demonstrated. A total of 1996 mini-, 4062 micro-, and 37 satellites motifs were identified. The microsatellites appeared widely and evenly spread over the whole genome sequence, whereas the minisatellites were not randomly distributed. Indeed, minisatellites were found to be associated with transposable elements clusters. Telomeres exhibited a specific sequence with a T(n)AG(n) signature. A comparison between the two available genome sequences of A. bisporus was also performed and sheds light on the genetic divergence between the two varieties. Beyond their role in genome structure, repeats provide a virtually endless source of molecular markers useful for genetic studies in this cultivated species.