Michael P. Challen

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.

A comparison of methods for successful triggering of gene silencing in Coprinus cinereus

Post-transcriptional gene-silencing methods (PTGS), including RNAi, are becoming increasingly pervasive in functional genomics. To advance analysis of the recently sequenced Coprinus cinereus genome, a high throughput gene silencing method is essential. We have exploited the GFP reporter gene to evaluate and quantify efficacy of three different silencing strategies. Modular constructs that encompassed antisense, untranslatable sense, and RNAi-mediating hairpin sequences, were transformed into a GFP-expressing host strain. Transformants exhibiting strong downregulation and partial suppression of GFP were recovered with all three constructs. Analyses of protein and transcriptional nucleic acids revealed that the antisense and hairpin sequences yielded similar levels of GFP suppression, and were both more efficient than untranslatable sense sequences. Our antisense vectors will expedite functional characterisation of C. cinereus and the modular nature of the constructs should permit exploitation of directional cDNA libraries for high throughput screening.

A phylogenetic reconstruction and emendation of Agaricus section Duploannulatae.

Agaricus section Duploannulatae comprises the group of species allied with A. bisporus and A. bitorquis. Disagreement exists in the literature regarding the composition of this group. We used DNA sequence data from the ITS segments of the nuclear ribosomal DNA region, in a sample of European and North American isolates, to identify characters shared by this group, to further delimit species-level taxa within the section, and to develop a phylogenetic hypothesis. Shared polymorphisms that suggest a natural limit for section Duploannulatae were found. ITS1 data were assessed using parsimony, distance and maximum likelihood methods of phylogeny. The section Duploannulatae comprised six robust clades. Five clades corresponded to well characterized species from the temperate Northern Hemisphere (A. bisporus, A. subfloccosus, A. bitorquis, A. vaporarius, A. cupressicola). The sixth clade encompassed an A. devoniensis complex. Species concepts, nomenclature, and relationships are discussed and compared with prior reports.

Evaluation of agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance.

There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.

Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus

A double-stranded (ds) RNA hairpin-mediated down-regulation system was developed for the cultivated mushroom Agaricus bisporus, and the role of the urea cycle enzyme argininosuccinate lyase (asl) in mushroom post-harvest development was investigated. Hairpin expression vectors were constructed to initiate down-regulation of asl and introduced into A. bisporus by Agrobacterium tumefaciens-mediated transformation. Transcripts of asl were significantly reduced (93.1 and 99.9%) in two transformants and hairpin vector transgene sequences were maintained throughout sporophore development. Single and multiple hairpin integration events were observed in Southern analysis. Transformants with down-regulated asl exhibited reduced yield and cap expansion during post-harvest sporophore development. There were no detectable differences in urea levels between the hairpin-transformed and control strains. This is the first report of reduced gene expression resulting from the introduction of dsRNA hairpins in A. bisporus and the applications of this technology will facilitate functional studies in the mushroom.

Agaricus section Xanthodermatei: a phylogenetic reconstruction with commentary on taxa

Agaricus section Xanthodermatei comprises a group of species allied to A. xanthodermus and generally characterized by basidiomata having phenolic odors, transiently yellowing discolorations in some parts of the basidiome, Schaeffer’s reaction negative, and mild to substantial toxicity. The section has a global distribution, while most included species have distributions restricted to regions of single continents. Using specimens and cultures from Europe, North America, and Hawaii, we analyzed DNA sequences from the ITS1+2 region of the nuclear rDNA to identify and characterize phylogenetically distinct entities and to construct a hypothesis of relationships, both among members of the section and with representative taxa from other sections of the genus. 61 sequences from affiliated taxa, plus 20 from six (or seven) other sections of Agaricus, and one Micropsalliota sequence, were evaluated under distance, maximum parsimony and maximum likelihood methods. We recognized 21 discrete entities in Xanthodermatei, including 14 established species and 7 new ones, three of which are described elsewhere. Four species from California, New Mexico, and France deserve further study before they are described. Type studies of American taxa are particularly emphasized, and a lectotype is designated for A. californicus. Section Xanthodermatei formed a single clade in most analyses, indicating that the traditional sectional characters noted above are good unifying characters that appear to have arisen only once within Agaricus. Deep divisions within the sequence-derived structure of the section could be interpreted as subsections in Xanthodermatei; however, various considerations led us to refrain from proposing new supraspecific taxa. The nearest neighbors of section Xanthodermatei are putatively in section Duploannulati.

Characterization of Serine Proteinase Expression in Agaricus bisporus and Coprinopsis cinerea by Using Green Fluorescent Protein and the A. bisporus SPR1 Promoter

ABSTRACT The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.

Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments.

The chromosomal region containing pab-1, mip, and the A mating type locus of the secondarily homothallic homobasidiomycete Coprinus bilanatus

Abstract In this paper we describe the cloning of the DNA region containing the A1 mating type genes of the secondarily homothallic mushroom Coprinus bilanatus and compare its organization to that of heterothallic homobasidiomycetes. As in other species, the C. bilanatus A factor contains several different genes that encode two different types of homeodomain transcription factor (HD1 and HD2); and some of these genes are active in the heterologous host C. cinereus. The HD1 and HD2 genes are distributed over two closely linked subloci, Aα and Aβ. A gene coding for a mitochondrial intermediate peptidase (mip) directly flanks the Aα sublocus. The pab-1 gene, required for para-aminobenzoic acid synthesis, is found 39 kb upstream of mip. The structural arrangement of this chromosomal region closely resembles the heterothallic C. cinereus. In contrast, the Aα and Aβ subloci of Schizophyllum commune are further separated, with pab-1 located between the two subloci, suggesting that a translocation event may have occurred during evolution.

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi.