ORIGINAL ARTICLE

Dikaryotic fruiting body development in a single dikaryonof Agrocybe aegerita and the spectrum of monokaryoticfruiting types in its monokaryotic progeny

Robert Herzog1,2,3 & Irina Solovyeva1,2,3 & Martin Rühl3,4 & Marco Thines2,3,5 &

Florian Hennicke1,3

Received: 16 January 2016 /Revised: 29 July 2016 /Accepted: 3 August 2016 /Published online: 5 September 2016# German Mycological Society and Springer-Verlag Berlin Heidelberg 2016

Abstract Using monokaryotic offspring from severaldikaryotic parental strains, the phenomenon of monokaryoticfruiting has been previously analysed in the commerciallycultivated high-quality edible mushroom Agrocybe aegerita,revealing a variety of monokaryotic fruiting types. Here, wereport a single dikaryotic A. aegerita strain, A. aegeritaAAE-3, and 40 monokaryons derived from it, which exhibit awide spectrum of monokaryotic fruiting types, including arare, previously unknown type. Advantageously, the selectedparental strain A. aegerita AAE-3 completes its life cyclewithin three weeks by the formation of dikaryotic fruitingbodies of typical agaric morphology on malt extract agarplates. In order to morphologically compare normal dikaryotic

fruiting to monokaryotic fruiting, histology was performedfrom all dikaryotic fruiting body development stages and allfruiting types of monokaryotic origin. No clamp connectionsor dikaryotic hyphae were observed within the plectenchymaof monokaryotic fruiting stages. Among the monokaryoticfruit ing types of the A. aegeri ta AAE-3-derivedmonokaryons, we also characterised the rare ‘stipe type’ heredescribed as ‘elongated initials type’ as no differentiation intoa future cap and stipe was seen. The two mating-compatiblemonokaryotic strains representing the extremes of the fruitingtype spectrum observed, A. aegerita AAE-3-13 (‘myceliumtype’) and A. aegerita AAE-3-32 (‘abortive + truehomokaryotic fruiting fruiter type, AHF+THF fruiter type’),were also found to readily produce oidia (arthrospores). Inorder to obtain a set of mating-compatible monokaryons cov-ering the whole observed spectrum of monokaryotic fruiting,the two monokaryons A. aegerita AAE-3-40 (‘initials type’)and A. aegerita AAE-3-37 (‘elongated initials type’) havebeen selected for their mating compatibility with A. aegeritaAAE-3-32 and A. aegerita AAE-3-13, respectively. Togetherwith the parental dikaryotic strain A. aegerita AAE-3, this setof standard monokaryons could prove useful for studies ex-ploring the factors regulating monokaryotic fruiting in com-parison to dikaryotic mushroom formation.

Keywords Agrocybe aegerita . Basidiomycota . Fruitingbody development . Mushrooms .Monokaryotic fruiting .

Morphogenesis

Introduction

Model organism-based research on fruiting body formation isa key to strain and breeding strategy optimisation in order toincrease quality and productivity in edible mushroom

Section Editor: Dominik Begerow

Robert Herzog and Irina Solovyeva contributed equally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s11557-016-1221-9) contains supplementary material,which is available to authorized users.

* Florian Hennickeflorian.hennicke@senckenberg.de

1 Junior Research Group Genetics and Genomics of Fungi,Senckenberg Gesellschaft für Naturforschung, Georg-Voigt-Str. 14–16, 60325 Frankfurt am Main, Germany

2 Institute of Ecology, Evolution and Diversity, Goethe-University,Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany

3 LOEWE Excellence Cluster for Integrative Fungal Research (IPF),Georg-Voigt-Str. 14–16, 60325 Frankfurt am Main, Germany

4 Institute of Food Chemistry and Food Biotechnology,Justus-Liebig-University Giessen, Heinrich-Buff-Ring 17,35392 Gießen, Germany

5 Senckenberg Biodiversity and Climate Research Centre (BiK-F),Senckenberg Gesellschaft für Naturforschung, Senckenberganlage25, 60325 Frankfurt am Main, Germany

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production (Kothe 2001). The genetic fundamentals offruiting body formation have been analysed repeatedly usingmodel fungi like Schizophyllum commune (Esser et al. 1979;Knabe et al. 2013; Ohm et al. 2010, 2011; Palmer and Horton2006; Pelkmans et al. 2016; Wessels 1993), Coprinopsiscinerea (Kamada 2002; Kamada et al. 2010; Stajich et al.2010; Kües 2000; Wälti et al. 2006) or Agrocybe aegerita(Esser and Meinhardt 1977; Labarère and Noël 1992;Meinhardt and Esser 1981; Meinhardt and Leslie 1982;Sirand-Pugnet et al. 2003; Wang et al. 2013), employing mi-crobiological, classical genetics and molecular biology meth-odologies. Not least, such research has yielded a more or lessdetailed picture of the different steps traversed during thefruiting body morphogenesis of those agaricomycete modelfungi. Even so, it still remains to be further studied in howfar certain developmental processes in these steps are homol-ogous or analogous between the different model agaric species(Kües and Navarro-González 2015). The model fungusA. aegerita (Sirand-Pugnet et al. 2003), also referred to asA. cylindracea (Uhart and Albertó 2007), is, furthermore, acommercially cultivated top-quality edible mushroom (Uhartet al. 2008) and its fruiting bodies have an excellent aromaprofile (Kleofas et al. 2014). In the course of studies onfruiting body formation in the mentioned model basidiomy-cetes including A. aegerita, the phenomenon of monokaryoticfruiting has also been studied, mainly employing classicalmicrobiology, biochemistry and genetics, rather than molecu-lar biology methodology (Esser et al. 1974, 1979; Esser andMeinhardt 1977; Labarère and Noël 1992; Leonard and Dick1968; Leslie and Leonard 1979; Meinhardt and Esser 1981;Miyake et al. 1980; Uno and Ishikawa 1971; Yli-Mattila et al.1989).

Esser et al. (1974), Esser and Meinhardt (1977) andMeinhardt and Esser (1981) employed three different parentaldikaryotic strains of A. aegerita, describing three major phe-notypes of monokaryotic fruiting exhibited by theirmonokaryotic progeny. Non-fruiting monokaryons were ofthe ‘mycelium type’ the ones only forming fruiting body ini-tials were assigned to the ‘initials type’while the ones produc-ing fully developed fruiting bodies were referred to as belong-ing to the ‘fruiter type’. Beyond that, within the monokaryoticoffspring from two out of fourteen dikaryotic strains of vari-ous geographic origins, i.e. in the offspring from the dikaryonsHU-3 and HU-4 (from Hurbanova, Czechoslovakia), a rareadditional type of monokaryotic fruiting, the ‘stipe(“Stielchen”) type’, has been reported once but not beenanalysed in detail in the dissertation of Meinhardt (1980).Since then, this monokaryotic fruiting type has not been stud-ied further. Interestingly, a similar spectrum of monokaryoticfruiting types seems to be present in Schizophyllum commune(Esser et al. 1979). In A. aegerita, Esser et al. (1974), Esserand Meinhardt (1977) and Meinhardt (1980) discerned asmaller size of monokaryotic fruiting bodies predominantly

bearing two-spored basidia in comparison to dikaryoticfruiting bodies bearing four-spored basidia. Labarère andNoël (1992) then subdivided the A. aegerita fruiter type intothree subgroups, the ‘abortive homokaryotic fruiting (AHF)’without cap-opening and basidiospore production, the ‘truehomokaryotic fruiting (THF)’ with reduced sporulation oftwo-spored basidia and the ‘pseudo-homokaryotic fruiting(PHF)’ with abundant sporulation of four-spored basidia.They also noticed that, in certain single monokaryons, evenmore than one of these subtypes of the fruiter type couldemerge from the same homokaryotic culture being AHF+THF or AHF+PHF. To indirectly check on the hyphal mor-phology within the three different subtypes of fruiting bodies,explants from AHF, THF and PHF fruiting bodies were made,which gave rise to mycelia without any clamp formation in thecase of explants from AHF and THF fruiting bodies, whileoutgrowing hyphae from PHF fruiting body explants exhibit-ed clamp connections. PHF was shown to result from matingtype switching, thus being dependent on different A and Bmating type alleles in the hyphae, and is, therefore, consideredequal to the normal dikaryotic fruiting, as it involves homo-dikaryotisation of primary homokaryons without plasmogamy(Labarère and Noël 1992).

In the present study, 40 monokaryotic strains of A. aegeritawere derived from the dikaryon A. aegerita AAE-3. Usingthem, we have, for the first time, histologically analysed thespectrum ofmonokaryotic fruiting inA. aegerita including theAHF+THF fruiter type of Labarère and Noël (1992) and thestipe type of Meinhardt (1980), which is here described as‘elongated initials type’. Two monokaryons displaying theextremes of the fruiting type spectrum, A. aegerita AAE-3-13 of the mycelium type and A. aegerita AAE-3-32 of theAHF+THF fruiter type, were selected for being mating-compatible and for readily producing oidia (arthrospores). Acomplete set of standard monokaryons representing the wholespectrum of monokaryotic fruiting was then obtained by mat-ing A. aegerita AAE-3-13 and A. aegerita AAE-3-32 with allother A. aegerita AAE-3-derived monokaryons of the initialstype and the elongated initials type.

Materials and methods

Strains, culture maintenance and fruiting induction

In this study, the A. aegerita dikaryotic parental strainA. aegerita AAE-3 was used for all experiments. Thisstrain is a derivate of the strain 4022 from Sylvan Inc.(Horst, The Netherlands), obtained by germinating a massof basidiospores on 2 % Malt Extract Agar (MEA; contain-ing 2 % malt extract [70167-500G, Sigma-Aldrich ChemieGmbH Munich, Germany], 1.5 % agar per L dH2O;autoclaved) into multiple monokaryons and picking a

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dikaryon resul t ing from a mating of compatiblemonokaryons. Forty A. aegeri ta AAE-3-der ivedmonokaryons, A. aegerita AAE-3-6 to A. aegerita AAE-3-45, were generated by single basidiospore isolation andgermination on MAT agar (prepared as in Grumbt et al.(2011), containing 0.2 % glucose, 0.1 % peptone, 0.1 %KH2PO4, 0.1 % MgSO4 and 1.6 % agar per L dH2O). Inbrief, for MAT preparation, 10 mL of a filter-sterilised(0.2 μM pore diameter, VWR International) 10 %KH2PO4 and 10 % MgSO4 stock are added to 880 mL ofautoclaved 1.6 % water agar supplemented by 100 mL ofseparately autoclaved Sabouraud medium [containing 2 %glucose, 1 % peptone, according to the composition ofSabouraud agar in McGinnis (1980), excluding agar].Monokaryons were confirmed to be truly monokaryoticby the absence of clamp connections and by 4 ′,6-diamidino-2-phenylindole (DAPI)/Calcofluor White stain-ing (for experimental details, see below). Agrocybeaegerita mycelia were routinely propagated on MEA at25 °C in the dark and stored as frozen stocks in distilledwater with 15 % glycerol at −80 °C. For routine propaga-tion of mycelia on agar plates, 2 % MEA was employed.For mating experiments, MAT agar plates were used. Forfruiting, 1.5 % MEA was used to limit the production ofaerial mycelium, so as to ease the recognition and pickingof individual dikaryotic or monokaryotic fruiting stages.Plates were centrally inoculated with a mycelium-overgrown agar plug of 0.5 cm2 diameter and first incubat-ed at 25 °C in the dark for 10 days (dikaryon) or 14 days(monokaryons), until mycelia reached the edge of theplates. Subsequently, plates were transferred to fruitingconditions into transparent non-air-tight lid-covered 70 %ethanol-disinfected wet chambers with saturated humidityand put into a 12-h white light/12-h darkness regime at20 °C using type 1501 Rumed growth chambers (RubarthApparate GmbH, Laatzen, Germany). Light intensity wasmeasured both with a Gigahertz Optik P9710 optometer(Gigahertz-Optik Vertriebsgesellschaft für technischeOptik mbH, Türkenfeld, Germany) and with a LI-250ALight Meter (LI-COR Bioscience, Bad Homburg,Germany). A medium light intensity of 1100 ± 100 luxand 7.9 μE± 0.5 μE m−2 s−1, respectively, was detected(light source: Osram Lumilux 5400 K A G13 daylightL36W/954-1 FLH, Osram AG, Munich, Germany).Aeration of the wet chambers was carried out in a laminarflow hood each day in the morning at the time the lightswitched on. In case of the monokaryons submitted tofruiting conditions, an agar plug of 0.5 cm2 diameter waspunched out and removed from the fully grown plates inorder to provide an additional local fruiting stimulus. Fromdays 21 to 23 post inoculation, mature fruiting bodiescould be observed with A. aegerita AAE-3 or on days 28to 30 for the monokaryotic strains of the fruiter type. Each

dikaryotic and monokaryotic fruiting experiment was re-peated at least three times independently, each comprisingat least three replicates.

Isolation and germination of basidiospores

Mature fruiting bodies of A. aegerita AAE-3 were pinned tothe lids of Petri dishes using aseptic vaseline. The lids werethen put over empty Petri dishes. If necessary, the stipe wasshortened using a scalpel. After 24 h, the accumulated spore-print of brown basidiospores in the bottom part of the Petridish was suspended in sterile water and plated in dilutionseries onto MAT agar and incubated at 25 °C. Single germi-nated mycelia were picked as soon as possible with a steriletungsten steel needle to prevent intergrowth or mating andseparated to fresh MAT plates. The monokaryotic state wasthen verified by the absence of clamps and DAPI/CalcofluorWhite staining.

Oidia induction conditions

Monokaryotic mycelia were placed centrally onto MAT agarplates (90 mmdiameter) on a single overgrown agar block andincubated at 25 °C in the dark for 14 days until plates werefully colonised. Plates were not sealed as aeration improvedthe production of aerial mycelium. Per plate, 5 mL dH2Owereadded onto the mycelium and the oidia rubbed off gently witha Drigalski spatula. The oidia suspension was then transferredinto an appropriate polypropylene tube, passing through a cellstrainer (40 μm pore diameter, Becton Dickinson GmbH,Heidelberg, Germany) to get rid of hyphal debris.Centrifugation at 6000×g for 5 min was sufficient to pelletoidia. Storage in water is possible for about a week at 8 °C butgermination is only slowed down by this. However, oidia canalso be suspended in 15 % glycerol and frozen at −80 °C forlong-term storage.

Mating experiments

Mating experiments were performed using MAT agar platesand micro-cultivation chambers to determine the mating com-patibility of different monokaryons. Micro-cultivation cham-bers were assembled as follows: the bottom of a Petri dish wascovered with sterile, wet filter paper. Then, two sterile micro-scope glass slides (76×26 mm), one put on top of the other,were placed on top of the paper. Finally, a 1.5 % MEA agarplug of 0.5 cm2 diameter overgrown by mycelium was inoc-ulated on the top glass and covered with a microscopecoverslip.

For mating, mycelia were inoculated about 1 cm apart fromeach other in the centre of a Petri dish (90 mm diameter, MATagar) or in the micro-cultivation chamber and incubated at25 °C in the dark until the two mycelia formed a large zone

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of contact (5–10 days, depending on the pair of monokaryoticstrains used). To analyse the mating interaction between twoindividual monokaryons, mycelia of both mating partnerswere first examined using light microscopy. The presence ofclamp connections on hyphae resulting from mating meantthat a dikaryon was formed and, therefore, monokaryons car-ried different alleles for the mating gene lociA and B (A≠, B≠).The dikaryotic state of a mycelium resulting from a compati-ble mating interaction was then further confirmed employingDAPI/Calcofluor White staining.

Microtome sections

A method used to prepare fruiting body specimens for micro-tome sectioning was published earlier (Knabe et al. 2013) andwas used here with some modifications. In brief, a specimenwas fixed in Pfeiffer’s solution (Pfeiffer 1898) and subse-quently dehydrated step-wise in methanol. The specimenwas then embedded into the cold-hardening resin Technovit7100 (Heraeus Kulzer GmbH, Hanau, Germany). Differingfrom the manufacturer’s instructions, the time of pre-infiltration was increased (1 to 2 days instead of 2 h).Microtome sections of 4–8 μm thickness were made using aRM 2155 rotary microtome (Leica Biosystems NusslochGmbH, Nussloch, Germany).

Sections were transferred to a microscopy slide onto a dropof dH2O. Sections fully expanded on the surface of the drop.The slides were then left to dry until the sections stuck tightlyto the glass and subsequently stained with toluidine blue Osolution (0.1 % toluidine blue [w/v], 0.1 % borax [w/v], 2 %sucrose [w/v] in dH2O) in a glass chamber for 20–40 s. Slideswere then destained in glass chambers filled with dH2O for20–30 min to reduce background staining and, afterwards,placed on paper cloth to dry. Finally, liquid Merckoglas(Merck, Darmstadt, Germany) was applied onto all sectionson each slide, then covered with a coverslip on top and left toharden for at least 2 h in order to obtain permanent microscopyslides. Bright field micrographs were taken with an AxioLab.A1 microscope (Carl Zeiss AG, Oberkochen, Germany)and Moticam 3.0 MP digital camera with Motic Images Plus2.0 software (Motic Deutschland GmbH, Wetzlar, Germany).Up to 162 micrographs of 200× or 100× magnification levelswere stitched seamlessly together to show complete fruitingbody stages using the ImageJ2 plugin ‘Image Stitching’(Preibisch et al. 2009).

Nuclear and cell wall staining, fluorescence microscopy

Micro-cultures on glass slides from the micro-cultivationchambers were stained without fixation. First, all remainingagar was removed from the glass slide. The remaining thinlayer of mycelium on the glass was covered with a 1.25 μg/mL solution (diluted in dH2O) of Calcofluor White (18909-

100ML-F, Sigma-Aldrich Chemie GmbH Munich, Germany)and incubated for 10 min. The slide was then briefly rinsed indH2O and the mycelium covered in a 4′,6-diamidino-2-phenylindole (DAPI, 6335.1, Carl Roth GmbH & Co. KG)embedding solution (Fischer and Timberlake 1995) for20 min, with a final DAPI working concentration of1.25 μg/mL (diluted in embedding solution). If necessary,the DAPI staining was repeated a second time. Selected tolu-idine blue O unstained microtome section slides from fruitingbody specimens were stained using the same protocol as withthe micro-cultures.

Oidia were centrifuged to a pellet at 12000×g for 5 minand the supernatant discarded. Then, the pellet was resuspend-ed and incubated in DAPI embedding solution for 20 min at25 °C, subsequently rinsed once with dH2O and resuspendedagain in dH2O. A drop of the oidia suspension was then ex-amined using fluorescence microscopy.

Microscopic examination was performed with an AxioImager.M2 microscope with DAPI filter (Carl Zeiss AG,Oberkochen, Germany). Micrographs were taken with anAxioCam MRc5 digital camera and Axiovision 4.6 software(Carl Zeiss AG, Oberkochen, Germany).

Results

Qualities of the selected dikaryotic strain A. aegeritaAAE-3 and its monokaryotic progeny

The selected dikaryon of the commercially cultivated mush-room A. aegerita, A. aegerita AAE-3, displays a ‘textbook’basidiomycete life cycle completed within three weeks by theformation of fruiting bodies of typical agaric morphologybearing basidiospores on four-spored basidia on MEA(Fig. 1). Agrocybe aegerita AAE-3-derived monokaryonsbearing one nucleus per hyphal segment (Fig. 2a) generatedfrom germinated single A. aegerita AAE-3 basidiospores canundergo mating with a compatible mating partner, yieldingstable dikaryotic hyphae with constant clamp formation(Fig. 2b). As especially monokaryotic mycelia of A. aegeritaare capable of oidia formation (Walther and Weiß 2006),A. aegerita AAE-3-derived monokaryons were tested for thisfeature. As a result, we selected a pair of mating-compatiblemonokaryons, i.e. the strains A. aegerita AAE-3-13 andA. aegerita AAE-3-32, both for displaying a rather intensiveoidia formation and for showing monokaryotic fruiting typesrepresentative for the extremes of the fruiting spectrum (seebelow). Oidia amounts in the order of 106 to 107 per fullycolonised 9 cm diameter MAT plate could be harvested whencopious aerial mycelium was formed by such a monokaryon.The monokaryotic state of the oidia was confirmed by nuclearand cell wall staining (Fig. 2c–d).

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Spectrum of monokaryotic fruiting typesin the AAE-3-derived monokaryons

With the 40 A. aegerita AAE-3-derived monokaryons, thethree major monokaryotic fruiting types from Esser et al.(1974), Esser and Meinhardt (1977) and Meinhardt andEsser (1981) could be observed, the mycelium type, theinitials type and the fruiter type. These types arecomplemented by an additional one only mentioned inthe dissertation of Meinhardt (1980), the ‘st ipe(“Stielchen”) type’, referred to as the elongated initialstype in this study (Fig. 3), as histology did not revealany differentiation into stipe and cap (see below).Macroscopica l ly, the e longated in i t ia l s type ischaracterised by an elongate plectenchymatic structure,which is brownish at the base and white at the apex.The different fruiting types of the individual A. aegeritaAAE-3-derived monokaryons are given in Table 1.

As a next step, a set of four standard monokaryons wasselected both representing the whole spectrum ofmonokaryotic fruiting and being mating-compatible withat least one other standard monokaryon. This wasachieved by mating A. aegerita AAE-3-13 (myceliumtype) and A. aegerita AAE-3-32 (fruiter type) with all

the A. aegerita AAE-3-derived monokaryons of the ini-tials type and the elongated initials type. As the matingreactions of A. aegerita are supposed to be controlled by atetrapolar mechanism of homogenic incompatibility in-volving two unlinked loci A and B (Meinhardt andLeslie 1982), clamp connections on hyphae resulting frommating indicated that a dikaryon was established carryingdifferent alleles for the mating gene loci A and B (A≠,B≠). Employing this criterion, we selected monokaryonA. aegerita AAE-3-37, belonging to the elongated initialstype, for its mating compatibility with A. aegerita AAE-3-13 and monokaryon A. aegerita AAE-3-40 of the initialstype for its compatibility with A. aegerita AAE-3-32(Fig. S1). Beyond that, the monokaryons A. aegeritaAAE-3-21 and A. aegerita AAE-3-28 of the initials typealso exhibited mating compatibility with A. aegeritaAAE-3-32 (data not shown).

Fig. 2 Visualisation of the nuclear state and septa allocation by nuclearand cell wall staining in dikaryotic and monokaryotic Agrocybe aegeritahyphal segments and oidia, respectively. The blue arrows indicate septa,while the red arrows indicate nuclei. Each scale bar represents 10 μm. aFluorescence microscopy photograph of a monokaryotic hyphal segmentfrom micro-cultivation chamber-grown mycelium of the A. aegeritamonokaryon A. aegerita AAE-3-16 with a single nucleus and simplesepta at its ends. b Fluorescence microscopy photograph of a dikaryotichyphal segment from micro-cultivation chamber-grown mycelium of thedikaryon A. aegerita AAE-3-2 ×A. aegerita AAE-3-6 with two nucleiand clamp connection-bearing septa at its ends. c Light microscopyphotograph (corresponding to the fluorescence microscopy photographin Fig. 2d) of monokaryotic oidia harvested from monokaryoticmycelium of A. aegerita AAE-3-13 grown on MAT agar after 14 daysat 25 °C in the dark. d Fluorescence microscopy photograph ofmonokaryotic oidia harvested from monokaryotic mycelium ofA. aegerita AAE-3-13 grown on MAT agar after 14 days at 25 °C inthe dark. The monokaryotic state of the oidia was visualised by nuclearand cell wall staining

Fig. 1 Tetrasporic basidia from a mature fruiting body of Agrocybeaegerita AAE-3. For fruiting body production, 1.5 % MEA was used.Centrally inoculated 1.5 % MEA plates were first grown at 25 °C in thedark for 10 days. Subsequently, fruiting conditions were applied(saturated humidity, 20 °C, a 12-h light/12-h darkness regime, aerationonce every day). Photographs were taken from toluidine blue O stainedmicrotome sections of a mature A. aegerita fruiting body. Each scale barrepresents 10 μm. a A basidium with four attached basidiospores. bBasidiospore tetrads as seen on the surface of a lamella from a maturefruiting body

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Histology of dikaryotic and monokaryotic fruiting bodymorphogenesis in A. aegeritaAAE-3 and its monokaryoticprogeny, respectively

The dikaryotic fruiting body formation of A. aegeritaAAE-3 takes place in four major steps. Fruiting bodyinitials (Fig. 4a–b) do not display much hyphal differ-entiation yet, in contrast to later stages of the fruiting

body development. Nevertheless, a basic first three-zonestructure can be distinguished, i.e. an inner lilac-stainedlower bulb, an upper blue-stained region and an outerless intensely blue-stained hyphal layer (Fig. 4b). Withinthe pr imordia (Fig . 4c) , fu ture cap and st ipeplectenchyma has become clearly recognisable. Also, atthe edge of the lower side of the future cap, the gillcavity has formed, where the hymenium is about to

Fig. 3 Monokaryotic fruiting ofAgrocybe aegerita AAE-3-derived monokaryons.Representative monokaryoticstrains displaying the four majormonokaryotic fruiting types‘mycelium type’ (mycelium),‘initials type’ (initials), ‘elongatedinitials type’ (elongated) and‘fruiter type’ (fruiter) were grownon 1.5 % MEA at 25 °C in thedark for 14 days. Subsequently,fruiting conditions were applied(saturated humidity, 20 °C, a 12-hlight/12-h darkness regime,aeration once every day).Photographs were taken at days28 to 30. Each scale bar represents0.5 cm

Table 1 Fruiting types of the individual Agrocybe aegerita AAE-3-derived monokaryons

‘Mycelium type’ ‘Initials type’ ‘Elongated initials type’ ‘Fruiter type’

A. aegerita AAE-3-13, -15,-17, -23, -24, -35, -42

A. aegerita AAE-3-6, -7, -9, -12, -18,-21, -25, -28, -29, -31, -40, -43, -44

A. aegerita AAE-3-14, -20,-22, -30, -36, -37, -38

A. aegerita AAE-3-8, -10, -11, -16, -19,-26, -27, -32, -33, -34, -39, -41, -45

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develop on the very early gill plectenchyma. In additionto these central plectenchyma parts, the outer veil of theprimordial cap and stipe can be recognised. Duringfruiting body maturation (Fig. 4d–e), several major de-velopments take place, i.e. stipe elongation, cap expan-sion, hymenium and gill generation plus development,as well as basidia and basidiospore formation (Fig. S2a–b). During this developmental step, the veil becomesthinned out on the outer surfaces of the immaturefruiting body (Fig. 4e) after it initially conspicuouslycovered the outer surfaces of the young mushroom(Fig. 4d). Fully developed fruiting bodies consist of acompletely elongated stipe and a totally expanded andopened cap, releasing high numbers of basidiosporesfrom fully grown gills (Fig. 4f). At this final stage ofdevelopment, part of the veil has remained macroscopi-cally visible as an annulus at the upper part of the stipe(Fig. S2c). Checking the nuclear state of hyphae withina mature dikaryotic A. aegerita fruiting body revealedthat they were composed of dikaryotic hyphal segmentswith simple septa at their ends (Fig. S2d). When depriv-ing A. aegerita AAE-3 initials of light, we could alsoinduce a ‘dark stipe’ phenotype (data not shown) well-known from other agarics, such as the model mushroomC. cinerea (Terashima et al. 2005).

Monokaryotic fruiting in the A. aegerita AAE-3-derived monokaryons was also analysed by histology.Microtome sections were made from all monokaryoticfruiting types displaying fruiting body developmentstages, i.e. from the initials type, from the elongatedinitials type and from the fruiter type (Fig. 5). Thiswas only performed for the final stage of fruiting bodydevelopment of each type analysed. Both fruiting bodyinitials of the initials type, from A. aegerita AAE-3-18(Fig. 5a, left picture), and initials of the elongated ini-tials type from strain A. aegerita AAE-3-22 (Fig. 5a,central picture), do not display a differentiation into dis-tinct plectenchyma parts relating to a primordial cap andstipe. Nonetheless, as also seen with the initials of thedikaryon A. aegerita AAE-3 (see Fig. 4a–b), a basicthree-zone structure can be distinguished in both typesof monokaryotic initials as well, i.e. an inner lilac-stained lower bulb, an upper blue-stained region andan outer a bit less intensely blue-stained hyphal layer(Fig. 5a, left and central pictures). Apart from beingelongated, we could not detect any other morphologicalconspicuities in the plectenchyma of the elongated ini-tials type. Fruiting bodies of the fruiter type fromA. aegerita AAE-3-32 (Fig. 5c, right picture; Fig. S3)resembled the dikaryotic fruiting bodies of A. aegerita

Fig. 4 Histology of the dikaryotic fruiting body formation of Agrocybeaegerita AAE-3. Mycelium of A. aegerita AAE-3 was grown on 1.5 %MEA at 25 °C in the dark for 14 days. Subsequently, fruiting conditionswere applied (saturated humidity, 20 °C, a 12-h light/12-h darknessregime, aeration every day). Photographs were taken from toluidineblue O stained microtome sections of the four major fruiting bodydevelopment stages of A. aegerita AAE-3. a Early fruiting body initialconsisting of not yet much differentiated hyphae in contrast to later stagesof the fruiting body development. The scale bar represents 100 μm. bLate fruiting body initial showing an already more condensedplectenchyma. The scale bar represents 100 μm. c Primordiumdisplaying the beginning of plectenchyma proportion differentiationinto future cap and stipe. The scale bar represents 200 μm. d Early

immature fruiting body exhibiting the initiation of hymenium and gillformation at the underpart of the developing cap plectenchymaproportion (still flat in profile, but already more intensely stained thansurrounding plectenchyma). The scale bar represents 200 μm. e Lateimmature fruiting body displaying stipe elongation, cap expansion andgill expansion in progress. Basidiospore formation is taking place at thesurface of the hymenium.Moreover, a secondary primordium can be seenat the basis of the stipe of the immature fruiting body. However, this is tobe seen as a rather exceptional coincidental event. The scale barrepresents 500 μm. f Fully developed fruiting body consisting of acompletely elongated stipe, a totally expanded plus opened cap andtotally unfolded gills containing multitudes of basidiospores at thesurface of the hymenium. The scale bar represents 1 mm

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AAE-3 (see Fig. 4d–f) in their vigorous growth andmorphology. Nevertheless, in such fruiting bodies, eithercap-opening and gill development were observed to beclearly reduced (Fig. 5c, right picture) or cap-openingwas seen to be still partially impaired (Fig. S3a). Inaddition, the former fruiting bodies (Fig. 5c, right pic-ture) were sterile, i.e. they did not produce any basid-iospores. The latter fruiting bodies (Fig. S3) showed alow basidiospore production, but only displaying two-spored basidia (Fig. S3c). Four-spored basidia, as theyare characteristic to normal dikaryotic fruiting bodies(see Fig. 1) could not be observed. Still, as seen duringfruiting body maturation of the dikaryotic fruiting bod-ies of A. aegerita AAE-3 (see Fig. 4d–e), the veil hasalso become thinned out on the outer surfaces of theA. aegerita AAE-3-32 fruiting bodies at their final stageof development (Fig. 5a, right picture; Fig. S3a).

Applying nuclear and cell wall staining and fluores-cence microscopy to selected toluidine blue O unstainedslides of fruiting stages from A. aegerita AAE-3-18,A. aegerita AAE-3-22 and A. aegerita AAE-3-32(Fig. 5b, from left to right), we could confirm that allmonokaryotic fruiting stages observed in this studyconsisted of monokaryotic hyphae without clamp connec-tions. In contrast to that, mature dikaryotic A. aegeritafruiting bodies displayed dikaryotic hyphae as it can bedetected from microtome sections, which were submittedto nuclear and cell wall staining (see Fig. S2d).

Discussion

Dikaryotic and monokaryotic fruiting bodymorphogenesis in A. aegerita

We have histologically monitored both the dikaryotic fruitingbody development of the parental dikaryon A. aegerita AAE-3 and all the monokaryotic fruiting types exhibited by theA. aegerita AAE-3-derived monokaryons. Starting from thisinitial analysis, developmental events taking place in bothdikaryotic and monokaryotic fruiting body development canbe further compared in future studies.

Generally, comparing both dikaryotic and monokaryoticfruiting, dikaryotic hyphal segments, present in dikaryoticfruiting stages, could not be observedwithin the plectenchymaof monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons. Plectologically, fruiting body initialsof strain A. aegerita AAE-3-18 of the initials type ofmonokaryotic fruiting do not seem to differ from dikaryoticfruiting body initials, except for not being constituted ofdikaryotic hyphae but monokaryotic ones. In contrast to that,apparently, the plectenchymatic structures observed withmonokaryon A. aegerita AAE-3-22 of the elongated initialstype do not have a morphological counterpart in the dikaryoticfruiting body development. Not displaying any differentiationinto plectenchyma proportions of a primordial cap and stipe,they also morphologically differ from the dark stipe pheno-type observed with dikaryotic fruiting body primordia

Fig. 5 Histological analysis of monokaryotic fruiting of themonokaryons Agrocybe aegerita AAE-3-18 (‘initials type’), A. aegeritaAAE-3-22 (‘elongated initials type’) and A. aegerita AAE-3-32 (‘fruitertype’). The monokaryons were grown on 1.5 %MEA at 25 °C in the darkfor 14 days. Subsequently, fruiting conditions were applied (saturatedhumidity, 20 °C, a 12-h light/12-h darkness regime, aeration every day).Photographs were taken from microtome sections of monokaryoticfruiting stages, which were harvested at days 28 to 30. a Photographsof toluidine blue O stained microtome sections from final fruiting stagesof monokaryons each exhibiting a certain type of monokaryotic fruiting.The scale bar represents 100 μm with A. aegerita AAE-3-18 and

A. aegerita AAE-3-22. With A. aegerita AAE-3-32, the scale barrepresents 1 mm. b Photographs of DAPI-/Calcofluor White-stainedmicrotome sections from fruiting stages of monokaryons eachexhibiting a certain type of monokaryotic fruiting. The image detailsshown by the photographs are localised at the transition area betweenthe upper core and the fringe of the fruiting structure. In the fruiter type,this corresponds to the transition area between the upper outer stipe andcap plectenchyma, which is not yet part of the veil (plectenchyma fillingthe gill cavity spanning between the edge of the gills and the surface of thestipe). The blue arrows indicate clamps, while the red arrows indicatenuclei. Each scale bar represents 10 μm

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incubated in continuous darkness, which at least show a dif-ferentiation into distinct plectenchyma proportions of a pri-mordial cap and stipe. Moreover, elongated initials show anintense brown colouring of their outer plectenchyma at thebase. Dikaryotic fruiting body development stages display auniform dark brown pileipellis colouring, especially beforematurity. This was also reported previously when producingfruiting bodies on wheat straw substrate (Uhart and Albertó2007). A uniform dark brown pileipellis colouring could alsobe seen in monokaryotic fruiting bodies of the fruiter type. Inmonokaryotic fruiting bodies of strain A. aegerita AAE-3-32(fruiter type), we could either observe a reduced cap-openingand gill development without basidiospore production or aslightly impaired cap-opening and the production of a fewbasidiospores on two-spored basidia. Thus, following the clas-sifications of Labarère and Noël (1992), the former fruitingbodies were assigned to the mode of abortive homokaryoticfruiting (AHF) without basidiospore production, while thelatter ones were assigned to the mode of true homokaryoticfruiting (THF) with some basidiospore production on two-spored basidia. AHF fruiting bodies of AAE-3-32 alsodisplayed a disproportionally flat cap as compared to imma-ture dikaryotic fruiting bodies of AAE-3 disposing of a moregibbous cap. This might be due to the reduced gill develop-ment observed with AHF fruiting bodies, which is not onlyincreasing in width but also in height during fruiting bodymaturation in normal dikaryotic fruiting bodies.

Analysing the capacities of different monokaryotic strainsto undergo monokaryotic fruiting, in S. commune, local injuryof the mycelium was previously found to be a trigger ofmonokaryotic fruiting to a certain extent (Leonard and Dick1973; Leslie and Leonard 1979). We were also curious to atleast briefly check this in monokaryotic fruiting of theA. aegerita AAE-3 progeny qualitatively. Therefore, we ap-plied a local injury stimulus to the mycelium of allmonokaryons by removing a circular agar plug from eachplate when submitting them to fruiting conditions. Leonardand Dick (1973) found that injury could work as a triggerfor monokaryotic fruiting, depending on both a sufficient de-gree of injury and on an interplay with other stimuli, such asthe density of the hyphae within the injured mycelial mass andthe nutrient level of the culture medium. Looking at the 40AAE-3-derived monokaryons under the applied fruiting con-ditions, we could see that there has often, but not always, beenan emergence of fruiting stages, especially at the point ofinjury, irrespective of the type of monokaryotic fruiting a cer-tain monokaryon exhibited, except for the ones of the myce-lium type (data not shown). In accordance to Leonard andDick (1973), we would expect that this tendency should in-crease in proportion to the intensity of the injury stimulusapplied to the mycelium of the A. aegerita monokaryons, aslong as a sufficient density of mycelium remains to be trig-gered to fruiting.

Monokaryotic fruiting of A. aegerita in comparisonto monokaryotic fruiting in other model basidiomycetes

According to Stahl and Esser (1976), monokaryotic fruitingappears to be a rather common phenomenon that has largelybeen neglected or dismissed as an abnormality. Accordingly,this phenomenon has been studied to a limited extent inagaricomycete fungi other than A. aegerita, including themodel mushrooms C. cinerea and S. commune (Esser et al.1979; Leonard and Dick 1968; Leslie and Leonard 1979;Miyake et al. 1980; Uno and Ishikawa 1971; Yli-Mattilaet al. 1989). Except for the ‘stromatic proliferations’ and ‘re-supinate hymenia’ of monokaryotic fruiting in Polyporusciliatus and the ‘stroma phenotype’ in S. commune, the spectraof monokaryotic fruiting types recorded for P. ciliatus (Stahland Esser 1976) and S. commune (Esser et al. 1979) moreclosely resemble the pattern we observed in the monokaryoticoffspring ofA. aegeritaAAE-3 than the monokaryotic fruitingpattern published for C. cinerea (Miyake et al. 1980; Uno andIshikawa 1971). In both species, P. ciliatus and S. commune,Esser et al. (1979) and Stahl and Esser (1976) also reported astipe type of monokaryotic fruiting, which we refer to as theelongated initials type in A. aegerita AAE-3-derivedmonokaryons. Nevertheless, in contrast to the elongated ini-tials in the A. aegerita AAE-3-derived monokaryons and thestipe type structures of S. commune (Esser et al. 1979), inP. ci l ia tus , hymenia were formed on top of theplectenchymatic structures of the stipe type containing mainlytwo-spored basidia (Stahl and Esser 1976). In P. ciliatus,monokaryotic mycelia and fruiting stages were confirmed toremain truly monokaryotic for the whole life cycle, i.e. myce-lial and fruiting stages hyphae were reported to never havingformed clamp connections and that they contained only onenucleus per cell all along. Accordingly, only one nucleus waspresent in each basidium, which, further on, only underwentmitosis once in the basidium and once again in each of the twospores followed by back-migration of two nuclei into the ba-sidium itself (Stahl and Esser 1976). In a basidium of amonokaryotic fruiter ofC. cinerea, the processes were slightlydifferent, as the two nuclei generated by mitosis underwentkaryogamy and meiosis, leading to a four-spored basidium(Miyake et al. 1980).

In A. aegerita, Labarère and Noël (1992) showed that mat-ing type switching happened in both mating type loci, leadingto a truly dikaryotic state in homokaryotic fruiting bodiesexhibiting the PHF phenotype. However, they did not directlyexamine the respective nuclear state within the plectenchymaof PHF fruiting bodies with four-spored basidia and clamps incontrast to the ones within fruiting bodies of the AHF modewithout basidia and of the THF mode with reduced sporula-tion of two-spored basidia by applying histology and nuclearstaining. Furthermore, some of the individual monokaryoticfruiter strains of Labarère and Noël (1992) were able to give

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rise to more than one mode of fruiter type fruiting, meaningAHF+PHF or AHF+THF, respectively. In case of the con-certed production of AHF and PHF fruiting bodies in some oftheir monokaryotic strains, AHF fruiting bodies always ap-peared before PHF fruiting bodies and they did not furtherdifferentiate. In contrast to that, there was no preferential ap-pearance order when AHF and THF co-emerged in certainmonokaryons of Labarère and Noël (1992). In our case, afterfour independent fructification experiments carried out fol-lowing mycelial sub-cultivation, our standard monokaryonof the fruiter type gave rise to both the THF and the AHFphenotype of Labarère and Noël (1992). It has to be left opento future studies as to whether certain cultivation conditionssuch as media composition or temperature could also have aninfluence on the prevalence of one or the other fruiter typephenotype a certain monokaryon like A. aegerita AAE-3-32(capable AHF and THF) can exhibit. Apart from that, in caseof potentially comparing the three subgroups of the fruitertype defined by Labarère and Noël (1992) by cell biologyand molecular biology approaches in the future, it would bemeaningful to clearly distinguish between dikaryon-like de-velopmental programmes depending onmating type pathwayssuch as PHF and apparently strictly monokaryoticprogrammes like AHF and THF.

Suitability of A. aegerita for model organism-basedresearch on mushroom morphogenesis

We have analysed the fruiting body development of both a setof mating-compatible A. aegerita sibling monokaryons ofeach monokaryotic fruiting type and of their parentaldikaryon. The selected parental strain and its monokaryoticprogeny exhibit a short ‘textbook’ basidiomycete life cycleeasy to reproduce on agar media, including the formation ofmonokaryotic oidia, of clamps on dikaryotic hyphae and oftypical agaric fruiting bodies. The produced A. aegerita strainscan now be used to apply genomics, functional genetics andglobal transcriptomics approaches to them in order to eluci-date the genetics of monokaryotic fruiting in A. aegerita.Furthermore, being of culinary value and economical impor-tance (Kleofas et al. 2014; Uhart et al. 2008), A. aegeritamushrooms do not undergo autolysis at maturity, in contrastto fruiting bodies of the model agaric C. cinerea (Kües 2000).Therefore, fruiting body maturation, harvest and post-harveststudies as well as yield-increase experiments can be carriedout withA. aegerita, as it has been done with the economicallyhighly important white button mushroom Agaricus bisporus(de Groot et al. 1998; Hammond and Nichols 1975; Straatsmaet al. 2013). Finally, strain A. aegerita AAE-3-32 exhibitingthe AHF and THF mode of monokaryotic fruiting appearsparticularly interesting for future research. On the one hand,one could possibly first-time scrutinise the commonalities anddifferences between normal dikaryotic and monokaryotic

fruiting body development without starting from a homo-dikaryotisation because of mating type switching as inA. aegerita PHF fruiter strains (Labarère and Noël 1992) orbecause of defects in the A and B loci (Kües 2000), permittingfull mating self-compatibility as in Amut Bmut homokaryonsof the model agaric C. cinerea. This self-compatibility of aC. cinerea Amut Bmut homokaryon leads to the formation ofhomo-dikaryotic hyphae with fairly regularly distributedfused clamps plus dikaryotic hyphal segments (rather in sub-merged than in aerial mycelium) and, finally, to fruiting bodyformation (Kües 2000). On the other hand, the low-sporulating AHF+THF fruiter strain A. aegerita AAE-3-32has potential application in breeding research focussing oncommercial mushroom farming. Such sporulation-deficientstrains are desired (Okuda et al. 2013), since allergies againstbasidiospores are an increasing problem in this business, suchas in the case of the oyster mushroom Pleurotus ostreatus andthe shiitake mushroom Lentinula edodes (Sánchez 2010).

In conclusion, we, thus, feel that A. aegerita probably de-serves reappraisal as a model basidiomycete to study severalaspects of mushroom developmental biology and evolution,as well as providing a model for targeted breeding.

Acknowledgments This work was supported by the LOEWEExcellence Cluster of Integrative Fungal Research (IPF).

Compliance with ethical standards

Conflict of interest All named authors have agreed to the publicationof this work and the manuscript does not infringe any other person’scopyright or property rights.

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