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.
School of Biological Sciences, Life Sciences Building, 24 Tyndall Avenue, University of Bristol, Bristol, BS8 1TQ, UK.|Department of Life Sciences, Institute of Technology, Sligo, Ash Lane, Sligo, Ireland.
Mary N
MN
Heneghan
School of Biological Sciences, Life Sciences Building, 24 Tyndall Avenue, University of Bristol, Bristol, BS8 1TQ, UK.
Claire
C
Burns
School of Biological Sciences, Life Sciences Building, 24 Tyndall Avenue, University of Bristol, Bristol, BS8 1TQ, UK.|Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, UK.
Ana M S B
AM
Costa
Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, UK.
Kerry S
KS
Burton
Warwick HRI, University of Warwick, Wellesbourne, Warwick, CV35 9EF, UK.
Michael P
MP
Challen
School of Biological Sciences, Life Sciences Building, 24 Tyndall Avenue, University of Bristol, Bristol, BS8 1TQ, UK.
Andy M
AM
Bailey
School of Biological Sciences, Life Sciences Building, 24 Tyndall Avenue, University of Bristol, Bristol, BS8 1TQ, UK.
Gary D
GD
Foster
D000222:Adaptation, Physiological; D000364:Agaricus / Q000201:enzymology*; D017753:Ecosystem; D018515:Plant Leaves / Q000382:microbiology; D057057:Serine Proteases / Q000378:metabolism*; D012987:Soil
D012987:Soil; D057057:Serine Proteases