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

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

Characterization of serine proteinase expression in agaricus bisporus and coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 Promoter

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 differentiatio

Functional analyses of <i>Agaricus bisporus </i>Serine Proteinase 1 (SPR1) reveals a role in utilisation of humic rich substrates and adaptation to the leaf-litter ecological niche

Agaricus bisporus is a secondary decomposer fungus and an excellent model for the adaptation, persistence and growth of fungi in humic‐rich environments such as soils of temperate woodland and pastures. The A. bisporus serine proteinase SPR1 is induced by humic acids and is highly expressed during growth on compost. Three Spr1 gene silencing cassettes were constructed around sense, antisense and non‐translatable‐stop strategies (pGRsensehph, pGRantihph and pGRstophph). Transformation of A. bisporus with these cassettes generated cultures showing a reduction in extracellular proteinase activity as demonstrated by the reduction, or abolition, of a clearing zone on plate‐based bioassays. These lines were then assessed by detailed enzyme assay, RT‐qPCR and fruiting. Serine proteinase activity in liquid cultures was reduced in 83% of transformants. RT‐qPCR showed reduced Spr1 mRNA levels in all transformants analysed, and these correlated with reduced enzyme activity. When fruiting was induced, highly‐silenced transformant AS5 failed to colonize the compost, whilst for those that did colonize the compost, 60% gave a reduction in mushroom yield. Transcriptional, biochemical and developmental observations, demonstrate that SPR1 has an important role in nutrient acquisition in compost and that SPR1 is a key enzyme in the adaptation of Agaricus to the humic‐rich ecological niche formed during biomass degradation

Epigenomic regulation of human T-cell leukemia virus by chromatin-insulator CTCF

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes an aggressive T-cell malignancy and a variety of inflammatory conditions. The integrated provirus includes a single binding site for the epigenomic insulator, CCCTC-binding protein (CTCF), but its function remains unclear. In the current study, a mutant virus was examined that eliminates the CTCF-binding site. The mutation did not disrupt the kinetics and levels of virus gene expression, or establishment of or reactivation from latency. However, the mutation disrupted the epigenetic barrier function, resulting in enhanced DNA CpG methylation downstream of the CTCF binding site on both strands of the integrated provirus and H3K4Me3, H3K36Me3, and H3K27Me3 chromatin modifications both up- and downstream of the site. A majority of clonal cell lines infected with wild type HTLV-1 exhibited increased plus strand gene expression with CTCF knockdown, while expression in mutant HTLV-1 clonal lines was unaffected. These findings indicate that CTCF binding regulates HTLV-1 gene expression, DNA and histone methylation in an integration site dependent fashion

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

Peer reviewe

The Origin of Multiple B Mating Specificities in Coprinus cinereus

Mushrooms, such as Coprinus cinereus, possess large families of pheromones and G-protein-coupled receptors that are sequestered at the B mating-type locus and whose function is to confer vast numbers of different mating types. This ability results from complex patterns of cognate and noncognate pheromone/receptor pairings, which potentially offer a unique insight into the molecular interaction between receptor and ligand. In this study we have identified many more members of these families by molecular analysis of strains collected worldwide. There are three groups of genes at each B locus. We have identified two alleles of group 1, five alleles of group 2, and seven alleles of group 3, encoding in total 14 different receptors and 29 different pheromones. The specificity of many newly identified alleles was determined by transformation analysis. One striking finding was that receptors fall into groups based on sequence homology but these do not correspond to the groups defined by position, indicating that complex evolutionary processes gave rise to the B loci. While additional allelic versions may occur in nature, the number of B specificities possible by combination of the alleles that we describe is 70, close to previous estimates based on population analysis

Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism

Coniothyrium minitans colonises and destroys the sclerotia of Sclerotinia sclerotiorum in nature exhibiting ecologically obligate mycoparasitism as its spores remain dormant in soil and only grow actively in the presence of the sclerotia. Molecular mechanisms underlying sclerotial mycoparasitism are poorly defined. We identified 251 unisequences representing genes preferentially expressed by C millitalls during sclerotial mycoparasitism, substantially increasing the molecular knowledge of this commercially important biocontrol agent. Genes associated with signalling and cellular communication, degradation of host cell walls and energy reserves, nutrient utilisation, detoxification and stress response were identified suggesting that C minitans employs a number of key processes during host colonisation. Several of these genes are novel to fungal-fungal interactions (e.g. PTH11-like GPCR and the ETP gene cluster). Secretin receptor-like GPCR and the TGF-beta signalling system have not yet been characterised in filamentous fungi. This study provides the basis for in-depth gene function analysis in sclerotial mycoparasitism. (c) 2007 Elsevier Inc. All rights reserved

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 as] 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. (C) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved