Functional analysis of Agaricus bisporus serine proteinase 1 reveals roles in utilization of humic rich substrates and adaptation to the leaf-litter ecological niche.

Mary N. Heneghan,Michael P. Challen,Andy M. Bailey,Ana M. S. B. Costa,Claire Burns,Gary D. Foster,Kerry S. Burton

doi:10.1111/1462-2920.13350

Mary N. Heneghan, Michael P. Challen + Show 5 more

Open Access

https://doi.org/10.1111/1462-2920.13350

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Abstract

Summary 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.

Highlights

SummaryAgaricus 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
We describe here the silencing of the Spr1 gene, the biochemical and molecular analysis of transformants and the monitoring of transformants through mushroom sporophore development
Transformation of A. bisporus with these Hph-Spr1 silencing cassettes resulted in the generation of 8 sense (S), 14 antisense (AS) and 17 stop (ST) stable transformants

Summary

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. Basidiomycetes are probably the ecologically most significant group of fungi, involved in the breakdown and chemical conversion of litter components (Dix and Webster, 1995) They constitute a major fraction of the living biomass and are responsible for efficient degradation of many recalcitrant organic compounds in soil litter and the humic layer (Steffen et al, 2007). Whilst A. bisporus is well known for its commercial value as a cultivated mushroom, it is often overlooked that this and many other closely related species inhabit woodland and permanent pasture across most temperate environments, and there may be a vital role for proteases such as SPR1 in nutrient acquisition from humic associated material in soils and compost. We describe here the silencing of the Spr gene, the biochemical and molecular analysis of transformants and the monitoring of transformants through mushroom sporophore development

Results

Discussion

Experimental procedures