Growth, Differentiation and Sexuality

406 H.A.B. Wösten and J.G.H. Wessels
D. Haemolysins
Aegerolysin of A. aegerita, which is probably encoded
by the Aa-pri1 gene of A. aegerita (Fernandez
Espinar and Labarère 1997; Berne et al. 2002),
and ostreolysin of P. ostreatus are acidic proteins
of about 16 kDa which are specifically expressed
in primordia and immature fruiting bodies (Berne
et al. 2002). Both proteins have haemolytic activity
at nanomolar concentrations. By aggregating in the
plasma membrane, ostreolysin can permeabilize
cell membranes of erythrocytes and artificial lipid
bilayers by forming pores of about 4 nm (Sepčić
et al. 2003, 2004). To form a transmembrane core
complex for cytolysis, the closely related, 17-kDa
pleurotolysin A assembles with the 59-kDa protein
pleurotolysin B of P. ostreatus (Sakurai et al.
2004; Tomita et al. 2004). Evidence indicates that
haemolysis works according to the colloid osmotic
mechanism, i.e. it can be prevented by the presence
of osmotically active solutes with a diameter
exceeding that of the pore. Permeabilization of
membranes by ostreolysin is inhibited by lysophospholipids
(Sepčić et al. 2003). These lipids are wellknown
signalling molecules involved in a variety
of processes (Corda et al. 2002), including differentiation
(Spiegel et al. 2002). Therefore, it was
suggested that ostreolysin has a role in cell signalling.
Sepčić et al. (2003) suggested that the level
of lysophopholipids in the cell could regulate membrane
binding and pore-forming ability of ostreolysin.
By this action, it may be involved in apoptosis
(Walser et al. 2003), now known to be involved
in development of the basidiomycete fruiting body
(see above, and Chap. 9, this volume). Notably,
no permeabilization was observed of membranes
made of total lipid extract from fruiting bodies of P.
ostreatus.Thesemembraneswerestronginhibitors
of haemolysis of erythrocytes. This suggests that
membranes of fruiting-body hyphae of P. ostreatus
contain lysophospholipids (Sepčić et al. 2003).
It remains to be established whether there are differences
in the amounts of these lipids in hyphae
making up the fruiting body, which is to be expected
if ostreolysin fulfils a role in apoptosis of
specific cells.
E. Oxidative Enzymes
A role for laccases in oxidative cross-linking
was first proposed for polypores (Bu’Lock 1967;
Bu’Lock and Walker 1967), which become pig-
mented and woody by oxidation of phenolic
compounds. Since then, intracellular and extracellular
laccase activity has been reported in
primorida and fruiting bodies of a number of
basidiomycetes such as S. commune (Leonard
and Phillips 1973; Phillips and Leonard 1976;
de Vries et al. 1986), L. edodes (Leatham and
Stahmann 1981; Leatham 1985; Zhao and Kwan
1999; Nagai et al. 2003) and Volvariella volvacea
(Chen et al. 2003, 2004a,b). This supports a role
for these enzymes in fruiting-body development,
for instance, by oxidative cross-linking of hyphae
in fruiting bodies (Leatham and Stahmann 1981;
Wessels et al. 1985). Alternatively, the deposition of
oxidized phenolics resulting from laccase activity
in the walls may increase surface hydrophobicity
and thus aid in the occurrence of emergent
growth. Unfortunately, these roles have still not
been verified, for instance, by gene inactivation.
The eln2-1 mutant of C. cinereus was isolated
in a screen for developmental mutants (Muraguchi
et al. 1999). This mutant is characterized by dumpy
fruiting-body primordia. Cell morphogenesis and
tissue organization in the primordial shaft are affected.
As a result, the mature fruiting bodies of this
strain have short stipes (Muraguchi and Kamada
2000). The eln2 gene is constitutively expressed and
encodes a novel type of cytochrome P450 enzyme.
These enzymes are involved in the oxidative, peroxidative
and reductive metabolism of numerous
compounds. A deletion of 18 amino acids at the
C terminus is the cause of the mutant phenotype.
Possibly, this deletion results in a modified mode of
catalytic activity. How can this explain the mutant
phenotype? Muraguchi and Kamada (2000) gave
three explanations. First, a changed catalytic activity
may produce a toxic compound which affects
development in the primordial shaft. Second, activity
of the truncated enzyme may not result in
a product which is normally instrumental in development.
Finally, the mutant enzyme may overproduce
a normal metabolite or produce superoxide
radicals. A disruption of the eln2 gene should establish
if this gene is indeed involved in fruiting-body
development.
Expression of cytochrome P450 genes has also
been shown in mushrooms of A. bisporus and L.
edodes (de Groot et al. 1997; Akiyama et al. 2002;
Hirano et al. 2004) Interestingly, the cytochrome
P450 genes of L. edodes, Le.cyp1 and Le.cyp2,were
shown to be differentially expressed. MRNA levels
were higher in primorida and in the stipe of the
premature fruiting body (Akiyama et al. 2002). The