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Fungi are among the most sexually active organisms in the world, with
a quite staggering sexual productivity. Early this century the pioneering
mycologist Reginald Buller calculated that a single giant puffball may contain
7 trillion spores, and that a single large shaggy ink cap contains enough
spores to stretch 41 miles end to end.

The ‘fruiting bodies’ that suddenly erupt from the earth are the visible
signs of sex among fungi. But there is more to a mushroom than meets the
eye. A fruit body is a specialised reproductive factory within which multitudes
of different sexual events are taking place . Researchers throughout the
world are now trying to identify the biochemical events that control the
development of reproductive fruit bodies, and to understand how the genes
that underlie the mating types required for sexual reproduction in heterothallic
fungi work at the molecular level.

Unlikely as it might seem, understanding the sex lives of fungi may
benefit us. Some of the chemical sex factors that fungi produce when they
initiate sexual reproduction appear to regulate the switch from an asexual
to a sexual growth phase, and the ability to control this transition has
many potential uses for agriculture and food production. We might use a
group of such chemical signalling compounds, known as sex morphogens, to
induce delicacies such as morels and truffles to fruit, or to increase the
yield of commercial mushrooms. Meanwhile, sex factors that block reproduction
in the fungi that cause diseases in plants might lead to biocontrol agents,
once synthetic analogues are developed for the agrochemicals industry.

A multitude of sex factors coordinate the sexual development of fungi.
These fungal aphrodisiacs fall into two main groups. First, in so-called
heterothallic fungi, sex factors mastermind the initial recognition and
attraction of compatible sexual partners. Since the 1920s, researchers have
detected various pheromone-like compounds, active at very low concentrations,
which trigger the development of sexual filaments or hyphae – the structures
that fuse in fungal sex. These compounds also cause the mutual attraction
of compatible fungal partners.

In one of the first studies in the late 1930s, John Raper at Harvard
University discovered that female hyphae of the water mould Achlya release
a hormone that induces male hyphae to form short branches bearing male gametes.
These so-called antheridia then release a complementary hormone that induces
further development of the female hyphae. The hormones, antheridiol and
oogoniol, were later identified as sterols. In other early studies, researchers
found terpenoid trisporic acids in the Mucorale pin moulds. These suppress
the formation of asexual spores and trigger the production of sexual structures,
with compatible partners having incomplete but complementary biosynthetic
pathways.

More recently, biologists have found complementary hormones parisin
and sirenin, produced by male and female gametes respectively, in the water
mould Allomyces. These account for the mutual attraction of male and female
hyphae. Male spores of the bread mould Neurospora crassa also produce a
substance that attracts female hyphae towards them, while the budding yeast
Saccharomyces cerevisiae releases mating-type specific chemicals, various
peptides, that trigger the clumping of cells, bringing compatible nuclei
together.

Current research suggests that the second main group of sex factors
trigger and coordinate sexual development once an interaction between compatible
hyphae has occurred. Such ‘sex morphogens’ may act either by triggering
sexual reproduction as a whole, in homothallic species , or by specifically
inducing the initial development of the fruit body, independent of sex.
These compounds are particularly interesting because they appear to control
the transition from a relatively undifferentiated form to a well-ordered,
three-dimensional structure. Understanding this process may well provide
insights into how shape is determined during the development of other multicellular
organisms – even humans.

In 1984, David Ingram and his colleagues in our laboratory at Cambridge
identified a lipid extract, SF, from mated cultures of the plant pathogen
Pyrenopeziza brassicae. This substance switched the fungus from an asexual
to sexual growth pattern. Adding SF to a culture of P. brassicae composed
of only one mating type blocks the formation of asexual spores and triggers
the initial stages of fruit body development. SF is currently being purified,
but recently a single component appeared to produce the same response as
the complex SF mixture.

In 1987 Sewell Champe and his colleagues at the Waksman Institute in
New Jersey made a similar discovery. They identified a series of ‘precocious
sexual inducing’ (Psi) factors from sexual reproducing cultures of Aspergillus
nidulans. These can suppress the formation of asexual spores and turn on
sexual development in vegetative cultures. The Psi factors have been identified
as a set of long chain fatty acids. At the same time two Japanese researchers,
Genshiro Kawai and Yonosuke lkeda of the Noda Institute purified a group
of lipids known as cerebrosides. These can trigger the formation of fruit
bodies in the Basi-diomycete Schizophyllum commune. In addition, in 1989
Graham Gooday and his colleagues at the University of Aberdeen discovered
that diffusible sex factors can switch on sexual morphogenesis in the dermatophyte
Arthrodema incurvatun. Other chemical factors, including linoleic acid and
cyclic AMP, have also been linked to fruiting in fungi.

Sex factors are clearly important in the control of sexual development,
but little is known about what controls their production. To try to find
out, molecular biologists are focusing on mating-type genes in heterothallic
fungi. These seem to be regulatory genes, functioning as ‘master switches’
to govern the outcome of sexual development. Sexually compatible nuclei
have the appropriate mating-type genes, which direct the production of different,
but complementary, polypeptides. Researchers now think that when compatible
hyphae fuse, these polypeptides may combine to form a novel hybrid that
goes on to bind to a particular bit of DNA in the nucleus. This binding
activates a number of target genes in a developmental cascade, leading to
changes in protein synthesis appropriate for sexual reproduction, including
the manufacture of sex factors.

The first studies of how mating-type genes work focused on the Ascomycete
budding yeast Saccharomyces cerevisiae. This has two mating types a and
&agr;, determined by mating type genes that were cloned in 1981. Ira Herskowitz
and others at the University of California School of Medicine later demonstrated
that when yeast cells fuse a hybrid regulatory protein is formed from the
union of the polypeptides coded by the a and &agr; mating-type genes. The hybrid
product switches off production of pheromones and other genes active only
in unfused, haploid nuclei, allowing further sexual development.

Then in 1988, American groups led by Louise Glass, and Robert Metzenberg
at the University of Wisconsin and Chuck Staben then at Stanford University
succeeded in cloning the mating-type regions from Neurospora crassa, which
has two mating types named a and A bearing very different genes. Researchers
now believe that these genes control both the incompatibility of similar
mating types and the development of fruit bodies in N. crassa; both genes
are required for production of fully fertile reproductive structures.

The most elaborate mating-type genes are found in the Basidiomycotina,
the mushrooms and toadstools, with most containing two different A and B
regions. These were thought to involve a total of four genes, but last year
Lorna Casselton’s team, now at the University of Oxford, reported that the
regions were likely to be far more complex, with at least seven genes identified
in the ink cap Coprinus cinereus. The function of these genes is unclear.
But Robert Ullrich and his colleagues at the University of Vermont, studying
mating-type regions in the basidiomycete Schizophyllum commune, reported
that the polypeptide products from some of the mating-type genes probably
regulate the action of other genes.

All this tells us quite a bit about the regulation of sex in fungi,
but leads to yet another question: how are the mating-type genes themselves
controlled? Various scientists have suggested that a range of environmental
stresses may activate these genes. Other phenomena such as interactions
between mitochondria from compatible hyphae, once they have fused, may also
be important (See ‘Sexual politics in the cell’, Alan Rayner and Ian Ross,
New ÐÓ°ÉÔ­´´, 30 March 1991). Researchers hope that a combination of the
‘inside-outside’ approach, taken by those working on mating-type genes,
and the ‘outside-inside’ approach, taken by those of us interested in sex
factors, will ultimately converge to explain how the switch from asexual
to sexual reproduction in fungi is controlled at a biochemical and molecular
level. In the end, a knowledge of the processes involved in the sexual development
of these relatively amenable organisms may give us insights into the even
more complex developmental processes going on in other animals such as ourselves.

Alison Ashby is a research fellow at Jesus College at the University
of Cambridge and Paul Dyer is a research assistant in the Department of
Life Sciences at the University of Nottingham.

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1: INSIDE THE MUSHROOM’S REPRODUCTIVE FACTORY

What does ‘sex’ mean to a fungus? Sexual reproduction involves the fusion
of haploid nuclei bearing only one set of chromosomes to form a diploid
nucleus or zygote with the full complement of paired chromosomes. This can
go on to produce new haploid nuclei by a series of cell divisions known
as meiosis. These basic facts about sex remain the same in all fungi, although
there are a range of different life cycles; in some species the predominant
life form is diploid, whereas most fungi are haploid during the vegetative
phase of growth. Sexual reproduction provides a new mix of genes giving
fungi an evolutionary advantage in changing environmental conditions.

Many fungi can also reproduce asexually, forming spores by mitosis,
without the need for sex, but these spores usually contain exactly the same
genetic information as the parent fungus and so do not generate variation.
Asexual reproduction has been likened to a man buying 100 tickets in a raffle,
only to find that they all have the same number. A sexual punter may be
able to afford fewer tickets, but at least they will all have different
numbers.

The fruiting bodies and sexual spores formed by many fungi are also
highly resistant to adverse environmental conditions, and can remain dormant
for long periods, waiting for better times. Sexual reproduction may also
lead to the repair of random genetic damage in nuclei as genes recombine.

Conditions have to be right before fungi undergo sex. Temperature, humidity,
light and nutrition must be favourable. Frequently fungi initiate sex when
they are stressed, provided that they have stored up enough nutrients during
vegetative growth.

Genetic conditions must also be conducive to sex. Some fungi are homothallic,
being able to have sex by themselves, with identical haploid nuclei fusing
to form a diploid zygote. Other heterothallic fungi require the presence
of a compatible partner. For sex to be successful, individuals must be of
different mating type. At its simplest, individuals may be + or – types.
But they may further act as ‘males’ and ‘females’, according to the contribution
they make towards the formation of reproductive structures, and hundreds
of different mating types exist in some mushrooms and toadstools. In heterothallic
fungi, the thread-like elements that constitute the vegetative body of a
fungus, the hyphae, must contain compatible haploid nuclei. These fuse together
as a prelude to sex, so that compatible nuclei within the hyphae themselves
fuse together. Often specialised structures are formed to bring about such
hyphal fusion. Compatible haploid nuclei within fused hyphae may then coalesce
immediately, or form a long-lived partnership before finally yielding to
sex, as happened in the Basidiomycotina, the mushrooms and toadstools.

Given favourable conditions for sex, fungal hyphae aggregate and differentiate
to form novel reproductive structures in the process of sexual morphogenesis.
This involves the development of two main hyphal systems. One is concerned
with the production of the tissues of the fruit body itself. The other,
more limited, system develops within the cushioned matrix of the growing
fruit body and is where true ‘sex’ takes place, with the fusion of haploid
nuclei followed by meiosis giving rise to sexual spores. The growth of both
hyphal systems is highly coordinated, to ensure that a characteristic fruit
body shape results, favourable for the dispersal of spores. Sexual spores
are eventually ejected from the fruit bodies, to be picked up by wind currents
and spread into new areas potentially favourable for growth.

* * *

2: HOW DOES A TOADSTOOL GET ITS SHAPE?

Mushrooms and toadstools (agarics), and bracket fungi are the most well-known
fungal fruit bodies. These come in a wide range of shapes and sizes, all
the better to disperse the spores they bear.

In stalked forms the spores are ejected from the cap, which is born
aloft to ensure that spores can be dispersed by passing air currents. Stalks
may be solid or hollow, but are invariably cylindrical. Although a girder
form would provide more stability, it would create greater aerodynamic disturbance,
blowing spores back onto the stalk. Stalks grow away from the pull of gravity
to ensure that gills hang vertically, so that the spores can be easily ejected.
Large agarics have relatively stout stalks, and small ones relatively slender
ones, because the load increases as the cube of the linear measurements.
So as a toadstool doubles in size, the cap volume (and hence its weight)
increases eight times, whereas the cross section of the stalk merely increases
four-fold. To compensate, stalks become relatively thicker as the fruit
body grows larger.

If you turn a toadstool upside down, you’ll notice that most have gills
radiating out from the stalk, often with intercalated gills at the edge.
But long-lived bracket fungi and Boletes such as the Penny Bun (used in
packet soups) have a sponge-like matrix of narrow tubes or pores from which
spores are ejected. This polypore structure makes more efficient use of
the area below the cap than a gill system would, with less wasted space.
However, polypores, unlike gills, cannot finely readjust to a vertical position
if a toadstool is slightly dislodged.

A fungus’s source of food also determines the size of its toadstools.
Large forms tend to be mycorrhizal, or wood decomposers, with access to
a rich source of nutrients. Smaller toadstools may be litter decomposers,
growing only on a single leaf or twig, and have conical tops and thin gills
to make the most of resources.

Agarics may be many different colours, but no one is sure why. Pigments
may protect against ultraviolet radiation, or warn predators of toxicity.
Yet brightly coloured Amanita species, deadly to humanity, are quite happily
eaten by rabbits and deer.