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.

Transcriptomic analysis of the interactions between Agaricus bisporus and Lecanicillium fungicola.

Agaricus bisporus is susceptible to a number of diseases, particularly those caused by fungi, with Lecanicillium fungicola being the most serious. Control of this disease is important for the security of crop production, however given the lack of knowledge about fungal-fungal interactions, such disease control is rather limited. Exploiting the recently released genome sequence of A. bisporus, here we report studies simultaneously investigating both the host and the pathogen, focussing on transcriptional changes associated with the cap spotting lesions typically seen in this interaction. Forward-suppressive subtractive hybridisation (SSH) analysis identified 68 A. bisporus unigenes induced during infection. Chitin deacetylase showed the strongest response, with almost 1000-fold up-regulation during infection, so was targeted for down-regulation by silencing to see if it was involved in defence against L. fungicola. Transgenic lines were made expressing hairpin RNAi constructs, however no changes in susceptibility to L. fungicola were observed. Amongst the other up-regulated genes there were none with readily apparent roles in resisting infection in this susceptible interaction. Reverse-SSH identified 72 unigenes from A. bisporus showing reduced expression, including two tyrosinases, several genes involved in nitrogen metabolism and a hydrophobin. The forward-SSH analysis of infected mushrooms also yielded 64 unigenes which were not of A. bisporus origin and thus derived from L. fungicola. An EST analysis of infection-mimicking conditions generated an additional 623 unigenes from L. fungicola including several oxidoreductases, cell wall degrading enzymes, ABC and MFS transporter proteins and various other genes believed to play roles in other pathosystems. Together, this analysis shows how both the pathogen and the host modify their gene expression during an infection-interaction, shedding some light on the disease process, although we note that some 40% of unigenes from both organisms encode hypothetical proteins with no ascribed function which highlights how much there is still to discover about this interaction.

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.

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.