Download Differential expression of genes under control of the

MicrObio/ogy (1995), 141,1281-1288
Printed in Great Britain
Differential expression of genes under control
of the mating-type genes in the secondary
mycelium of Schizophyllum commune
Sigridur A. Asgeirsdbttir, Marianne A. van Wetter and
Joseph G. H. Wessels
Author for correspondence: Joseph G. H. Wessels. Tel: +31 50 632322. Fax: +31 50 632273.
e-mail : [email protected]
Department of Plant
Biology, Groningen
Biomolecular Sciences and
Biotechnology Institute
(GBB), University of
Groningen, Kerklaan 30,
9751 NN Haren, The
Netherlands
The Schizophy//umcommune SC3 gene, which encodes a hydrophobin that
coats aerial hyphae, is expressed in both monokaryons and dikaryons. The
dikaryons were formed by mating two monokaryons with different MATA and
MATB genes, leading to activation of the MATA- and MAT&controlled
pathways (MATA-on and MATBlon). In contrast to the monokaryons, the
dikaryons also expressed other hydrophobin genes (SCf, S U ) as well as a gene
(SC7) encoding a hydrophilic wall protein. None of these four genes was
expressed in MATA-off MATB-on mycelia, indicating that MA7B-on represses
SC3 and that both MATA-onand MA7B-n are required for activation of SCf,
SC4 and SC7. In fruiting dikaryons, immunolabelling revealed that SC3p was
produced by aerial hyphae but not by hyphae that constitute the fruit-body
tissue. In contrast to aerial hyphae, the latter produced dikaryon-specific
transcripts and secreted SC7p into the extracellular matrix of the tissue. This
suggests that in the aerial hyphae of the dikaryon the MATB-on pathway was
not effective (MATBloff). We observed that in these aerial hyphae the two
nuclei were wider apart than in a typical dikaryon. Although other
explanations are not ruled out, we tentatively propose that effective
interaction of different MATB genes requires proximity of the two nuclei
containing these genes, and that disruption of this binucleate state represents
a novel mechanism of gene control for spatial cell differentiation in the
secondary mycelium.
Keywords : SchixopLylium commune, mating-type genes, hydrophobin regulation, fruitbody formation
INTRODUCTION
Sexual morphogenesis in the homobasidiomycete Schixopbyllum commune is primarily controlled by multi-allelic
mating-type genes within the MATA and M A T B loci
(also called A- and B-incompatibility genes or incompatibility factors ; Raper, 1966, 1988). Mating of monokaryons (one nucleus per hyphal compartment) that
contain different M A T A and M A T B genes produces a
dikaryon (with clamp connections and two closely
associated nuclei per hyphal compartment). The monokaryon normally produces aerial hyphae only but the
dikaryon also forms multicellular fruit bodies in which
Abbreviations: DAPI, 4’,6-diamidine-2-phenylindole dihydrochloride;
WDF, polyvinylidenedifluoride; PVP, polyvinylpyrrolidone.
0001-9711 8 1995 SGM
haploid nuclei fuse to form diploid nuclei that immediately
undergo meiosis with the production of basidiospores.
Several MATAa loci of S. commune have been cloned and
sequenced (Specht etal., 1992; Stankis e t al., 1992). These
loci mostly contain allelic gene pairs with each of the two
different genes in a pair containing a particular homeoboxlike sequence, HDI or HD2. It appears that in a heterokaryon with different MATA genes ( M A T A + ) the
effective interaction ( M ATA-on) is between the product
of an H D I gene in one nucleus and the product of an HD2
gene in the other nucleus. Because of the presence of
homeobox-like sequences these products are thought to
be transcription factors that, in the effective combination,
regulate downstream genes involved in sexual morphogenesis, as discussed by Casselton & Kiies (1994). Less is
known about the structure and operation of the M A T B
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S. A. ASGEIRSDOTTIR a n d OTHERS
loci. These loci appear to contain genes for pheromones
and pheromone-receptors (Wendland & Kothe, 1994),
and thus would resemble the a mating-type genes of the
heterobasidiomycete U.rti/agomqdiis (Bolker e t a/., 1992).
Genes that are direct or indirect targets for regulation by
the mating-type genes of S. cornmane have been cloned
(Mulder & Wessels, 1986). The sequences of five of these,
belonging to two different gene families and encoding
abundant mRNAs, have been reported. The SC7/SC14
family encodes dikaryon-specific hydrophilic proteins
with homology to pathogenesis-related proteins (PR1
proteins) of plants (Schuren e t al., 1993). The
SCl/SC3/SC4 gene family encodes cysteine-rich hydrophobic proteins (Schuren & Wessels, 1990) which we
called hydrophobins (Wessels e t a/., 1991a, b). All five
proteins contain signal sequences for secretion and the
mature proteins encoded by SC7, SC3 and SC4 have been
found in the culture medium and associated with cell walls
in aerial structures. Except for the SC3 gene, which is also
active in the monokaryon, these genes are expressed to
high levels only in the dikaryon (MATA-on MATB-on)
(Mulder & Wessels, 1986; Ruiters e t a/., 1988). They are
all relatively inactive in young cultures that do, however,
express the monokaryotic and dikaryotic cellular phenotypes. These genes are thus unlikely to play a role in the
monokaryon-dikaryon transition. Their activation, as
shown by nuclear run-on transcription assays (Schuren e t
a/., 1993c), coincides with the formation of emergent
structures, aerial hyphae in the monokaryon and aerial
hyphae plus fruit-bodies in the dikaryon.
The SC3 hydrophobin self-assembles at the surface of
aerial hyphae into a so-called rodlet layer that is highly
insoluble and responsible for the high hydrophobicity of
the hyphal surface (Wosten e t a/., 1993, 1994a). Since SC3
is activated in both monokaryons (MATA-off M A T B ofF) and dikaryons (MATA-on MATB-on) it has been
suggested (Wessels, 1992) that this gene is not regulated
by the mating-type genes. However, in this paper we
demonstrate that in M A TA-off M A TB-on mycelia,
which form few aerial hyphae, the SC3 gene is downregulated, suggesting that this may also be the case in
M A TA-on M A TB-on hyphae. Using antibodies, we
found that in the fruiting M A T A + M A T B $ = heterokaryon hyphae within fruit bodies produce the dikaryotic
protein SC7p but not SC3p. On the other hand, aerial
hyphae formed on this mycelium do produce SC3p, but
the dikaryotic state appeared disrupted in the sense that
the nuclei were wider apart than in typical dikaryotic
hyphae and no clamp connections were formed. We
suggest that in these aerial hyphae the M A TB-on pathway
is inoperative, despite the presence of different M A T B
genes, allowing for formation of SC3p but not SC7p.
METHODS
Strains and culture conditions. The following strains of 5'.
commune were used in these experiments : the co-isogenic strains
4-39 (MATA41 MATB41, CBS 341.81) and 4-40 (MATA43
MATB43, CBS 340.81); a derived dikaryon; a heterokaryon
derived from a hemicompatible mating ( M ATA41M A TB41x
M A TA41 M A TB4,) and a co-isogenic mutant homokaryon
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with constitutive M A TB activity ( M ATA41 M A TB""). For
RNA isolation, the mycelia were grown for 4 d at 24 "C in the
light on minimal medium (Dons e t al., 1979) solidified with
0.7 YOagar. For immunodetection of SC3p and SC7p, dikaryons
were grown for 5 d from a central inoculum on 1.5 % (w/v) agar
minimal medium in a circulating air incubator in darkness at
24 OC and 95 YO relative humidity. The cultures were then
transferred to the light and incubated for an additional 48 h to
induce a ring of fruit-body primordia (Ruiters & Wessels, 1989).
When analysing the secretion of SC3p and SC7p by submerged
hyphae, the cultures were grown on perforated polycarbonate
membranes (standard pore size 0.1 pm; Poretics) overlying the
agar medium. After 5 d growth in the dark and 48 h in the light,
the colonies were transferred to a new Petri dish and proteins
were caught on a polyvinylidene difluoride (PVDF) membrane
positioned under the polycarbonate membrane for 6 h (Wosten
e t al., 1994a). For isolation of emergent hyphae, dikaryons were
grown for 2 d from a mycelial homogenate mixed with 9 vols of
3.3 YOlow-melting-point agarose layered on 3 % agar minimal
medium. The cultures were grown at 30 OC in the dark to
stimulate formation of aerial hyphae and to suppress formation
of fruit bodies.
RNA isolation and Northern blot analysis. Total RNA was
isolated by the hot-phenol procedure (Wessels e t al., 1987;
Schuren etal., 1993b). For Northern blot analysis, the RNA was
separated on agarose gels containing 2.2 M formaldehyde and
blotted onto Hybond N' membrane (Amersham) in 10 x SSC
(SSC is 150 mM sodium chloride, 15 mM sodium citrate,
pH 7.0). Blots were hybridized at 65 "C according to the method
of Church & Gilbert (1984) to ,,P-labelled cDNA clones
(Mulder & Wessels, 1986; Ruiters et al., 1988).
Isolation of emergent aerial hyphae and analysis of nuclear
distribution. Hyphae just emerging from a 3 ?LO low-meltingpoint agarose layer mixed with a mycelial homogenate, layered
on 3 % agar minimal medium, were isolated as follows. The
culture was fixed for 2 h at room temperature under vacuum in
freshly made 2.5 % (v/v) paraformaldehyde, 0.5 % glutaraldehyde, 0.1 M cacodylate buffer, pH 7.2. The culture was
washed thoroughly with water for 24 h to remove all fixative
from the plate and 5 ml 18 % gelatin solution (30 "C) was
poured onto the plate which was then stored at 4 OC overnight.
The gelatin layer containing emergent hyphae was carefully
peeled off from the plate, heated at 55 OC and hyphae were
centrifuged down in a Sorvall swing-out HB4 rotor at 1OOOOg
for 5 min. Hyphae were washed three times with pre-warmed
(50 "C) water, mounted on a microscope slide coated with
chrome-alum-gelatin (Kodak), stained with 0.5 pg ml-' DAPI
(4',6-diamidine-2-phenylindole dihydrochloride ; Boehringer
Mannheim) in 42.5 % (v/v) glycerol, 0.1 M Tris/HCl, pH 7.4
for 60 min at 37 "C, and fluorescence examined with a Zeiss
Axiophot microscope (filter combination BP 365, FT 395, LP
397).
lmmunodetection of SC3p and SC7p in aerial structures. For
immunodetection of SC3p and SC7p in differentiated aerial
structures, a fruiting dikaryotic colony was fixed in 4 % (w/v)
paraformaldehyde in phosphate-buffered saline (PBS is 137 mM
NaC1, 2.7 mM KC1, 1.6 mM NaH,PO,, 8-3mM Na,HPO,,
pH 7.6) according to the method of Ruiters & Wessels (1989).
Sections (25-30 pm) were cut perpendicular to the surface on a
freeze microtome and mounted on microscope slides coated
with chrome-alum-gelatin. Sections were blocked overnight at
room temperature in PBS containing 1 ?LO (w/v) BSA, 1 ?LO
(w/v) polyvinylpyrrolidone (PVP), 0.5 YOnormal goat serum,
0.03 % Tween 20,0*02YONaN, and 0-35 M NaC1. The antisera
used were raised against purified SC3p (Wosten e t al., 1994a) and
against a protein A-SC7p fusion protein produced in E. coli
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Cell differentiation in ScbiXoplyIIz4m commune
(Schuren e t al., 1993a). Prior to use, these antisera were purified
by incubation with isolated hyphal walls of a thn mutant as
described by Wosten e t al. (1994a), to prevent non-specific
reactions with cell-wall components. The purified sera were
diluted in blocking buffer (1 :500 and 1: 100 for anti-SC3p serum
and anti-SC7p serum, respectively) and incubated with the
sections at room temperature for 24 h. After four 5 min
washings with PBS supplemented with 0.35 M NaCl, the
sections were incubated for 2 h with goat-anti-rabbit antibodies
conjugated with 5 nm gold particles (Zymed) diluted in
blocking buffer according to the manufacturer's protocol. The
sections were washed as above and the immunogold labelling
was enhanced with silver (SilvEnhance-LM kit, Zymed).
2
lmmunodetection of SC3p and SC7p secreted into the culture
medium. PVDF membranes on which secreted proteins were
caught were blocked for 2 h at room temperature with 5 %
skimmed milk and 1 % PVP in PBS and then incubated for 1 h
with antisera against SC3p (diluted 1 :2000 in the blocking
buffer) or SC7p (dilution 1:200). The immunoreaction was
detected with goat-anti-rabbit IgG coupled with alkaline
phosphatase and using BCIP and NBT as substrates (Harlow &
Lane, 1988).
Electron microscopy. For low-temperature scanning electron
microscopy, agar blocks (approximately 5 x 5 mm) containing
fruit-body primordia were frozen in a mixture of solid and
liquid nitrogen. Primordia were broken with a cooled knife
within a Fisons E7400 cry0 unit, etched for 4 min and coated
with gold. Samples were examined at 20 kV in a Jeol JSM-35C
scanning electron microscope. Immuno-electron microscopy of
thin sections was performed according to Wosten et a/. (1994a),
except that primordia were fixed in 3 % paraformaldehyde,
0 1 M cacodylate buffer pH 7.2 and embedded in Unicryl
(BioCell).
3
RESULTS
SC3 is suppressed by the activity of the B mating-type
genes
Mycelia of S. commune in which only the MATB-on
pathway is operative, i.e. a MATA = M A T B heterokaryon (common-MA TA heterokaryon) and a MATA41
MATBco" homokaryon (in which a mutation constitutively activates the M A TB-on pathway), produce few
aerial hyphae (Fig. l a ) ; they have a so-called 'flat'
phenotype (Papazian, 1950; Parag, 1962). Because of the
known correlation between low SC3 expression and the
absence of aerial hyphae (Wessels et al., 1991b), the level
of SC3 m R N A in these mycelia was determined. Northern
blot analysis (Fig. lb) showed a high level of SC3 m R N A
in the monokaryon (lane l), but no hybridization signals
were detected with R N A from the MATBcon homokaryon and the common-MA TA heterokaryon (lanes 2
and 3), with similar amounts of RNA loaded on the gels
(lanes 4-6). This suggests negative regulation of SC3 by
the MATB-on pathway. Since a MATA+ M A T B +
heterokaryon does produce SC3 m R N A with the formation of aerial hyphae (Mulder & Wessels, 1986;Ruiters
e t al., 1988), this indicates that in these mycelia either
MA TA-on counteracts the suppressive effect of the
MATB-on pathway on SC3 activity, or that these mycelia
contain specialized hyphae in which the MATB-on
pathway is not operative.
(
b
1
nt
1
2
3
4
5
6
nt
+
4 1900
775 +
Fig. 1. (a) Morphology of 4-d-old cultures of the monokaryon
MATA41MA7B41(l), the homokaryon MATA41MA7Bcon(2), and
the heterokaryon MATA4' MA7B41 MA7B43 (3). (b) Northern
blot analysis of RNA of the cultures shown in (a) probed
respectively with SC3 cDNA (lanes 1-3) and 185 rDNA (lanes
4-61.
Localization of the secretion of SC3p and SC7p by
submerged hyphae
Submerged hyphae do not accumulate SC3p and SC7p in
their cell walls but secrete them into the culture medium
(Wessels e t al., 1991a, b; Schuren e t al., 1993a). The
secretion of these proteins in a fruiting MATA+
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S. A. ASGEIRSDOTTIR a n d OTHERS
+
heterokaryon was localized by immunodetection on a PVDF membrane placed under the colony.
Cultures were grown for 5 d in darkness and transferred
to light to induce the formation of a ring of fruit-body
primordia. The dikaryon-specific SC7p was primarily
detected in the zone of leading hyphae and in the zone
where fruit-body primordia were formed, whereas SC3p
was hardly detected in the fruiting zone but it was secreted
in the zone of leading hyphae and in the inner zone where
aerial hyphae were being formed (Fig. 2). These results
indicate heterogeneity of hyphae within the colony with
respect to expression of the SC3 and SC7 genes.
M A TB
lmmunolocalizationof SC3p and SC7p in emergent
structures
................................... ............................................................. ........................ ..................................
Fig, 2. lmmunolocalization of secretion of SC3p and SC7p a t the
colony level.A fruiting MATA* MA7B* heterokaQ'on
(a)
secretes SC7p in the zone of fruit-body formation and the zone
of peripheral hyphae (b), whereas SC3p is absent from the zone
containing fruit-body primordia but is secreted in central parts
of the colony forming aerial hyphae (c).
Immunolocalization of SC3p and SC7p on transverse
sections through a colony of a fruiting dikaryon (Fig. 3)
shows that SC3p, but not SC7p, was present in individually growing aerial hyphae emerging from the
substrate, and in hyphae covering the fruit body, and also
in the hymenium* However, hyphae making up the inner
tissue (PlectenchYma) of the fruit body did not Produce
SC3p but did react with SC7p antibodies.
Fig. 3. lmmunolocalization of SC7p (a, b) and SC3p (c, d) in fruit bodies (a, c) and aerial hyphae (b, d) of the MATA*
MA7B heterokaryon. Bars, 100 pm.
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Cell differentiation in Scbi~opbyllzimcommzine
phology in that they mostly contained simple septa or
pseudoclamps, i.e. clamp cells that have failed to fuse with
the penultimate cell. This is particularly clear in the long
hyphae that cover fruit-body primordia. These hyphae
were divided into several compartments by simple septa.
Since formation of clamp connections is closely associated
with the dikaryotic state of the M A T A + M A T B +
heterokaryon, we attempted to analyse the nuclear distribution. However, we were unable to stain nuclei in fullgrown aerial hyphae. Either the DAPI stain did not penetrate these thick-walled hyphae or the hyphae had lost
their nuclei. Giemsa or Feulgen staining was also unsuccessful. However, aerial hyphae just emerging from the
substrate mycelium, isolated by embedding them in
gelatin and peeling them off the surface, did contain
stainable nuclei. When distances between the perimeters
of stained nuclei were measured (see Fig. 5b), it was found
that these aerial hyphae (cell lengths 92*2+35-5pm)
contained two nuclei positioned a considerable distance
from each other (8.3 f 4.8 pm, minimum 1.4 pm, maximum 20.1 pm) in the 51 cells that were counted (Fig. 5b).
This is in contrast to the dikaryotic hyphae in the substrate
mycelium where the two nuclei occur in close proximity
(distance 0 9 k 0 . 4 pm; Fig. 5a). For comparison we
examined the nuclear distribution in a M A T A +
M A TB heterokaryon homozygous for the f17f mutation. In this mycelium, formation of all dikaryon-specific
transcripts is suppressed, no fruit bodies are formed but
SC3is highly expressed with abundant formation of aerial
hyphae (Springer & Wessels, 1989). The mutation also
prevents clamp formation by preventing the clamp cell
fusing with the penultimate cell (Springer & Wessels,
1989). As a consequence, only the apical cell is binucleate,
but all other cells are mononucleate with one nucleus
caught in the unfused clamp cell (Fig. 5c).
+
Fig. 4. (a) Low-temperature scanning electron micrograph of a
broken fruit body showing the loose contact between aerial
hyphae covering the fruit body and the extensive extracellular
matrix surrounding and binding together the hyphae of the
plectenchyma; bar, 100 pm. (b) lmmunolocalization of SC7p in
thin sections of the plectenchyma. The 15 nm gold particles
indicate the presence of SC7p within the extracellular matrix
(no signal was obtained with pre-immune serum or with the
anti-SC3p serum); bar, 0.5 pm.
Fig. 4 (a) shows the morphology of a developing fruit
body by cryo-scanning electron microscopy. Whereas the
outside of the fruit body is covered by individually
growing aerial hyphae that express SC3, the interior
hyphae that form the plectenchyma and express SC7 are
held together by an extensive extracellular matrix. Fig. 4
(b) demonstrates that SC7p is a constituent of this
extracellular matrix.
Nuclear distribution in aerial hyphae
+
+
Aerial hyphae of the M A TA M A T B heterokaryon
were found to deviate from the normal dikaryotic mor-
DISCUSSION
Recently we showed that the SC3gene of S. commune codes
for a hydrophobin that self-assembles at the surface of
aerial hyphae forming a highly insoluble hydrophobic
rodlet layer (Wosten et al., 1993, 1994a). A targeted
mutation in this gene (Wosten e t al., 1994b) reduces
formation of aerial hyphae under certain conditions,
indicating a primary role of SC3p in the formation of aerial
hyphae (unpublished observations). The tbn mutation that
inactivates all known hydrophobin genes, as well as other
genes involved in emergent growth, suppresses formation
of both aerial hyphae and fruit bodies (Wessels e t al.,
1991b). Another instance in which few aerial hyphae are
produced is when the M A TB-on pathway is operating in
the absence of the MATA-on pathway (Papazian, 1950;
Parag, 1962), producing a so-called flat mycelium (Fig.
la). In an earlier study (Ruiters e t al., 1988) we found that
in one such mycelium, the MATA41 MATBcon homokaryon, SC3 is not expressed. In the present paper we
show that this is also the case in a M A TA = MA 723
heterokaryon, indicating that the M A TB-on pathway
negatively regulates the expression of SC3 and thereby
suppresses formation of aerial hyphae.
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S. A. ASGEIRSDOTTIR a n d OTHERS
........................................................ ........................................................................................................................................................................................................................ ..............
Fig. 5. Nuclear positions in a typical dikaryotic hypha (a) and a hypha (b) just emerging from the substrate to become
aerial, both from the same MATA+ MATBP heterokaryon. Distances between nuclei were measured as indicated in (b).
Nuclear distribution in a MATA+ MATB 9 heterokaryon homozygous for fbf is shown in (c). Note the binucleate apical cell
at the top and the single nuclei caught in unfused clamp cells. Bar, 5 pm.
.......................................
~
Notwithstanding the repressing effect of the M A TB-on
pathway on expression of SC3, this gene is expressed in
fruiting MA TA
M ATB
heterokaryons and
MA TACOnM A TBcon homokaryons along with typical
dikaryotic transcripts which, in addition, require the
M A TA-on pathway for their formation (Mulder &
Wessels, 1986; Ruiters et a/., 1988). One explanation for
the (variable) expression of SC3 in these mycelia is that the
MA TA-on pathway counteracts the effect of the M A 2%on pathway on the activity of this gene. However, the
results reported in this paper show that there is a spatial
difference in the presence of SC3p and the typically
dikaryotic protein SC7p, both with respect to the sites of
secretion into the medium and the presence in emergent
structures; SC3p was only detected in aerial hyphae and
hymenium while the main tissue of the fruit body
produces SC7p but not SC3p. This agrees with the earlier
observation that in fruit bodies all dikaryon-specific
transcripts are abundant, but that the SC3 transcript is
low compared to other parts of the mycelium (Mulder &
Wessels, 1986), indicating that with respect to matingtype gene regulation, cell differentiation within the
M A T A + M A T B + mycelium has to be taken into
account.
+
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+
+
+
Normally, hyphae produced by the M A T A M A T B
heterokaryon have the dikaryotic morphology, i.e. each
cell contains two closely associated nuclei of opposite
mating type and a clamp connection at each septum.
Cytological evidence suggests that some structural material is holding the two juxtaposed nuclei together (Todd
& Aylmore, 1985). However, in aerial hyphae that
produce SC3p we found that this binucleate state is
disrupted and that nuclei occur at variable distances from
each other while, during continued growth of these
hyphae, clamp connections were rarely seen ; pseudoclamps and simple septa were abundant. This suggests
that in truly dikaryotic hyphae, such as in the fruit-body
plectenchyma, the M A TB-on pathway, together with the
M A TA-on pathway, results in formation of dikaryonspecific transcripts, but that the effective M A TB-on
pathway results in inactivity of the SC3 gene. We further
suggest that in emerging aerial hyphae the M A T B - o n
pathway is somehow switched off, resulting in activation
of SC3 and inactivation of genes producing dikaryonspecific transcripts.
How is the MATB-on pathway inactivated? The activity
of SC3 is correlated with the failure of clamp cells to fuse.
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Cell differentiation in Scbixoph_yllumcommune
In aerial hyphae this may result from the fact that diffusible
factors involved in fusion, possibly pheromones produced
under direction of the M A T B genes, are unable to reach
their targets in the air and therefore inactivation of the
MATB-on pathway occurs. Why this should lead to a
breakdown of the binucleate state in the aerial hyphae is
unclear because nuclear conjugation is considered a
function of the MATA-on pathway (Raper, 1966). In the
M A TA M A TB $. pf/pfheterokaryon, the absence of
some gene product apparently blocks clamp-cell fusion.
However, it is unlikely that this mutation directly
interferes with the activity or the interaction of the
M A TB genes since nuclear migration, another process
regulated by the M A T B genes, normally occurs in
matings of strains both carrying ?.$Another line of
reasoning, which we prefer, concentrates on the
significance of the binucleate state for proper interactions
between mating-type genes. Even in many basidiomycete
species that do not produce clamp connections, this
binucleate state is a most characteristic feature of the
secondary mycelium (Kiihner, 1977; Ross e t al., 1991). In
both the incipient aerial hyphae and thepfmutant of S.
commune the binucleate state is disrupted and we hypothesize that this leads to a shut-down of the MATB-on
pathway and expression in the secondary mycelium of a
phenotype similar to that of the primary mycelium.
+
If disruption of the binucleate state is responsible for the
expression of SC3, then this process must also regularly
occur in hyphae of the M A TA M A T B heterokaryon
growing submerged because SC3p is secreted under these
conditions (Wessels etal., 1991a). As shown in Fig. 2, The
SC3p-producing hyphae are not the same as those producing dikaryon-specific proteins. Microscopic observations show the regular occurrence of disruption of the
binucleate state (data not shown), but correlation with
SC3p production has not yet been investigated. Why SC3p
is also produced by hymenial cells is unclear, but these
cells obviously deviate from the binucleate state on their
way to diploidy and meiosis.
+
+
It is difficult to say whether the disruption of the
binucleate state also shuts down the MATA-on pathway.
A diagnostic feature of this pathway is the occurrence of
non-fused clamp cells (pseudoclamps) which were often
observed in aerial hyphae. However, one would not
expect a loss of interaction between M A TA genes to lead
to an immediate cessation of the MATA-on phenotype
because experimental de-dikaryotization has shown that
pseudoclamp formation can continue for many cell
divisions in derived homokaryons (Wessels e t al., 1976).
Although this is the first time that disruption of the
binucleate state has been related to developmental gene
regulation, disruption of the binucleate state has previously been observed in the secondary mycelium of
several basidiomycetes, leading to a phenotype called a
pseudo-primary mycelium (Kuhner, 1977). Gabriel (1967,
cited in Kiihner, 1977) found in Panellu-s ser0tinu.r and
Phlebia radiata that the factor responsible for the transition
to pseudo-primary mycelium is carbon dioxide accumu-
lation. Interestingly, in the dikaryon of S. commune,
increased carbon dioxide concentration leads to an
increase in transcripts from the SC3gene and to a decrease
in transcripts of the dikaryon-expressed SC genes, while
aerial hyphae become more abundant and formation of
fruit bodies is inhibited (Wessels e t al., 1987). It would,
therefore, not be surprising if in S. commune, too, carbon
dioxide has an effect on the binucleate state of the
secondary mycelium. Another example of disruption of
the binucleate state was presented by Ross e t a!. (1991),
who showed that an arginine-requiring mutant dikaryon
of C0prinu.r congregatus reversibly switched to the homokaryotic phenotype when deprived of exogenous arginine.
The phenomenon of phenotypic switching on the basis of
association-dissociation of nuclei in a hetero karyon,
affecting interaction of mating-type genes, may thus be of
general occurrence in basidiomycetes and may present a
mechanism for regulation of cell differentiation unique to
the fungi which mostly produce heterokaryons, rather
than diploids, after mating. How nuclear associationdissociation is regulated and how it affects the interactions
between different M A T B genes is unknown, but it may
become clear once the mechanism of action of these genes
is established.
ACKNOWLEDGEMENTS
We would like to thank J. Zagers and K. Sjollema for help with
the electron microscopy and J. M. J. Scheer for general
technical assistance.
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Received 22 December 1994; revised 16 February 1995; accepted
20 February 1995.
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