Functional analysis of Agaricus bisporus serine proteinase 1 reveals roles in utilization 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.

Improvement of the Coprinopsis cinerea molecular toolkit using new construct design and additional marker genes.

This paper describes the optimisation of an existing basidiomycete molecular toolkit through the development of new versatile vectors. These vectors enable the straightforward and rapid construction of gene expression and silencing cassettes by allowing the easy exchange of promoters, coding regions and terminator elements. The constructs contain multiple cloning sites (MCS) allowing any gene to be inserted using a range of restriction sites, with the option of a 5' integral intron for efficient gene expression. We describe the testing of these vectors through marker gene expression in Coprinopsis cinerea. This work also extends the range of marker genes available for use in C. cinerea with the first report of DsRed and monomeric red fluorescent protein (mRFP) expression in C. cinerea and further demonstrates the requirement for an intron in the expression cassette for some marker genes. However, analysis of transformants containing either beta-glucuronidase (GUS) or luciferase (LUC) genes, with and without an intron revealed no detectable marker gene expression. The inclusion of an intron does therefore not guarantee expression and other genetic factors may be involved.

Patients with painful bladder syndrome have altered response to thermal stimuli and catastrophic reaction to painful experiences.

AIMS: To compare cutaneous sensory thresholds, habituation to somatic stimuli, and tendency towards catastrophic reaction to painful stimuli in patients with Painful Bladder Syndrome (PBS) to controls without PBS. METHOD: Thermal and vibratory sensory thresholds were established in 11 PBS patients and 10 controls at C5, T1, T12, and S3 dermatomes. Supra-threshold thermal stimuli were then applied at T12 and S3 for 60 sec while patients periodically rated the intensity of stimuli using a visual analog scale. A Pain Catastrophizing Scale (PCS) questionnaire was also completed by all participants before testing. RESULTS: PBS patients were less sensitive to warm stimuli in the T12 dermatome than asymptomatic controls (thresholds 36.6 +/- 1.10 degrees C vs. 35.3 +/- 1.0 degrees C, P < 0.02) but otherwise had similar thermal and vibratory thresholds. Habituation to supra-threshold stimuli at T12 and S3 dermatomes was more common in controls than PBS subjects (7 (70%) vs. 2 (18%), P < 0.03 and 9 (90%) vs. 3 (27%), P < 0.008, respectively). The PCS score correlated with the duration of PBS symptoms and with thresholds to warm stimuli at T12 dermatome (rho = 0.65, P < 0.03 and rho = 0.5, P < 0.021, respectively). CONCLUSION: Our data suggests that habituation to stimuli may be impaired and that a catastrophic reaction to perceived stimuli may be involved in the sensory experience of PBS patients and facilitate chronic pain. Neurourol. Urodyn. 28:400-404, 2009. (c) 2009 Wiley-Liss, Inc.

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 differentiation.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase.

The tenS gene encoding tenellin synthetase (TENS), a 4239-residue polyketide synthase nonribosomal-peptide synthetase (PKS-NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M-2-3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C-terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans-acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.

The pOT and pLOB vector systems: improving ease of transgene expression in Botrytis cinerea.

This paper outlines the construction of a novel vector system comprising interchangeable terminators, as well as a multiple cloning site (MCS), to facilitate the transformation of the fungal plant pathogen Botrytis cinerea. Previous molecular studies on B. cinerea have relied upon the pLOB1 based vector system (controlled by the Aspergillus nidulans oliC promoter and a region reported to be the B. cinerea tubA terminator). Investigations, however, have revealed that, rather than the genuine B. cinerea tubA terminator, the pLOB1 terminator fragment is from another gene locus within the genome. Because previous studies have found that terminators aide in transcript stability, the main aims of this study were to develop and evaluate both vector systems, pOT (controlled by the A. nidulans oliC promoter and A. nidulans trpC terminator) and pLOB, with a range of exogenous genes, including enhanced green fluorescent protein (eGFP), monomeric red fluorescent protein (mRFP), luciferase (LUC) and beta-glucuronidase (GUS). Our investigations demonstrate that pLOB and pOT based vectors are capable of expressing all four reporter genes and may be applied to future molecular studies on B. cinerea and other related ascomycetes. Additionally, this is the first reported expression of mRFP and LUC in B. cinerea.

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.