The transcriptional regulator Emphasis Type="Ital

Authors: Jordi F Pelkmans Aurin M Vos Karin Scholtmeijer Ed Hendrix Johan J P Baars Thies Gehrmann Marcel J T Reinders Luis G Lugones Han A B Wösten

Publish Date: 2016/05/21

Volume: 100, Issue: 16, Pages: 7151-7159

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Abstract

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation Its inactivation results in a strain that is arrested at the stage of aggregate formation In this study the c2h2 orthologue of Agaricus bisporus was overexpressed in this white button mushroom forming basidiomycete using Agrobacteriummediated transformation Morphology cap expansion rate and total number and biomass of mushrooms were not affected by overexpression of c2h2 However yield per day of the c2h2 overexpression strains peaked 1 day earlier These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development making its encoding gene a target for breeding of commercial mushroom strainsThe basidiomycete Agaricus bisporus is cultivated globally for the production of white button mushrooms These fruiting bodies have a relative high protein content and contain fibers vitamins minerals and bioactive compounds A bisporus is grown on compost formed from wheat straw horse or chicken manure and gypsum During colonization the compost is topped with a casing layer needed for moisture and microbial flora BelsKoning 1950 Flegg 1956 Kalberer 1987 Induction of mushroom formation depends on environmental signals The volatile 1octen3ol represses early development while high temperature ie 25 °C instead of 18 °C inhibits development from smooth to elongated primordia On the other hand CO2 impacts the number of fruiting bodies that are formed Noble et al 2009 Eastwood et al 2013 Development of A bisporus is a complex process Kües and NavarroGonzález 2015 It starts with aggregation of hyphae into hyphal knots Umar and van Griensven 1997 These structures develop into 1–2mm initials also called primordia that differentiate by forming cap and stem tissues Umar and van Griensven 1997 Up to 10  of differentiated primordia develop into mushrooms Noble et al 2003 Breaking of the veil of these fruiting bodies enables airborne dispersal of basidiospores that had been formed in the gill tissue within the capProduction conditions of white button mushrooms have been optimized with respect to yield and quality of fruiting bodies Straatsma et al 2013 However molecular mechanisms underlying mushroom formation are poorly understood For instance transcription factors TFs involved in white button development have not been identified so far Such regulatory proteins have been identified in the model organism Schizophyllum commune Ohm et al 2010 2011 2013 Formation of its fruiting bodies is induced by blue light and is repressed by high CO2 Perkins and Gordon 1969 Niederpruem 1963 Raudaskoski and Viitanen 1982 Ohm et al 2013 The blue light receptor complex consists of the sensor WC1 and TF WC2 Inactivation of wc1 and/or wc2 results in a blind strain not able to produce aggregates primordia and fruiting bodies Ohm et al 2013 Strains in which the homeodomain gene hom2 or the zinc finger TF gene fst4 have been inactivated are also not able to produce aggregates Ohm et al 2010 2011 In contrast inactivation of the gene encoding the Cys2His2 zinc finger protein C2H2 results in a strain that does form aggregates but primordia and fruiting bodies are not formed Ohm et al 2011 Strains in which genes are inactivated that encode the zinc finger protein Fst3 the GATA type zinc finger protein Gat1 or the homeodomain protein Hom1 form smaller fruiting bodies but in higher numbers Ohm et al 2011 These proteins were proposed to play a role in repression of outgrowth of primordia into fruiting bodies or to play a role in expansion of the fruiting bodyHomologs of the S commune TFs involved in fruiting body development have been identified in other mushroomforming fungi Expression analysis in A bisporus Laccaria bicolor and Coprinopsis cinerea suggests that mushroom development in the Basidiomycota follows a core regulatory program with speciesspecific variations that explain differences in morphology and sensitivity to environmental signals Ohm et al 2010 Morin et al 2012 Plaza et al 2014 Muraguchi et al 2015 In this study the A bisporus c2h2 homolog was overexpressed in the commercial A15 strain of this mushroomforming fungus This resulted in an accelerated rate of mushroom production Experimental data indicate that C2H2 functions both early and late in mushroom development and that it is an interesting target for breeding of commercial strainsThe heterokaryotic A bisporus strain A15 obtained from the fungal collection of Plant Breeding Wageningen UR the Netherlands and its derivatives AT2731 and AT2735 that overexpress c2h2 were routinely grown at 25 °C on malt extract agar medium MEA 20 g l−1 malt extract agar BD biosciences Franklin Lakes USA 21 g l−1 MOPS pH 70 Spawn substrate was produced by heating 75 g of Sorghum seeds in water at 100 °C for 20 min after which 24 g kg−1 CaSO4 and 687 g kg−1 CaCO3 were added Spawn was colonized for 3 weeks at 25 °C using two 1weekold A bisporus colonies as inoculum Mushrooms were produced by inoculating boxes 40 cm width × 60 cm length × 22 cm height containing 16 kg phase 2 compost CNC Milsbeek The Netherlands with 75 g of spawn Compost temperature was maintained at 25 °C with an air temperature of 22 °C Relative humidity in growth cells was kept at 95  while CO2 levels fluctuated between 1500 and 2000 ppm Ten boxes were inoculated per strain and were randomly distributed in the growth cell After 16 days the compost in each box was topped with 7kg casing layer CNC Milsbeek The Netherlands Growth was prolonged for 14 days before venting The casing was manually broken 4 days prior to venting and mixed to create fast regenerative growth and a more equal distribution of A bisporus in the casing layer Venting resulted in a gradual decrease of compost and air temperature to 19 and 18 °C respectively Relative humidity and CO2 levels decreased gradually to 85  and 1200 ppm respectively The first buttons were removed from the bed 9 days after ventingPhotos of casing layer surfaces were taken in a fixed rig at 24h intervals from venting until the start of the first flush Emergence of mushrooms and growth rate of the caps were monitored using ImageJ http//imagejnihgov/ij/ Harvesting of mushrooms was done by a professional picker as performed in commercial production Prior to the flushes some buttons were removed to open up the space between developing buttons Fruiting bodies with a diameter between 40 and 60 mm were always harvested while fruiting bodies with a diameter of ≤40 mm were picked from densely populated areas to provide more space water and nutrients to the remaining mushrooms thereby ensuring optimal yield Mushrooms were classified as size 40 mushrooms with a cap ≤40 mm or size 60 mushrooms with a cap between 40 and 60 mm Mushrooms were harvested in two flushes All mushrooms had reached a size ≥40 mm during the second flush at day 22 and all fruiting bodies were therefore harvested thus completing the experiment Yield per box was expressed as the biomass and the number of harvested mushrooms Height and width of cap and stem were determined of 10 randomly selected mushrooms per box during the peak day of the first flush Dry weight of the mushrooms was assessed by drying 200 g wet weight fruiting bodies at 100 °C Relative dry weight is defined as the dry weight compared to the original wet weight

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