Fungi Page 5
The fruit bodies of ascomycetes, like those of basidiomycetes, are sexual organs that release spores that result from sexual interactions between mycelia of compatible mating type. Ascospores and basidiospores are sexual spores. The life cycles of many of these fungi—particularly the ascomycetes—are complicated by the production of other types of spores without any sexual process. These asexual spores, or conidia, are produced by single colonies and this mechanism of asexual reproduction is a very successful way for spreading the fungus (Figure 14). Depending upon food availability and other environmental conditions, and the proximity of compatible mates, a single ascomycete species can cycle between asexual reproduction using conidia and sexual reproduction using ascospores. This is called pleomorphism or pleomorphy. It creates problems for describing fungal species because some fungi have been named twice, once for the conidium phase, and a second time for the ascospore phase. This has resulted in the proliferation of Latin names for fungi and taxonomists try to cancel duplicate names when they are identified. We will consider other problems in taxonomy shortly.
14. Asexual spores (conidia) of a Penicillium species produced from a cluster of elongated cells at the top of a stalk (conidiophore).
The other major groups of fungi are less well known. They contain fewer described species than the basidiomycetes and ascomycetes, and few of them are visible without a microscope. Phylum Glomeromycota is a group of fungi that forms supportive relationships with plants called arbuscular mycorrhizas. The term ‘arbuscular’, meaning ‘dwarf tree’, refers to the finely branched structures that the fungus forms inside the root cells of its plant partner. Arbuscules provide a large surface area for the exchange of nutrients and water: the fungus supplies the plant with water and dissolved minerals and the plant provides the fungus with some of the sugar that it produces by photosynthesis. Zygomycete fungi used to be classified in a separate phylum, but recent research shows that these microorganisms are scattered across the evolutionary tree. They do not form a coherent natural group. Mating between two strains of the zygomycete Mucor is often used as a textbook example of a simple form sexual reproduction. Pilobolus (Figure 6) and Spirodactylon (Figure 8) are also zygomycetes. Other zygomycete fungi are important in food spoilage, are used to ferment soybean to produce tempe, and can cause serious human infections called mucormycoses.
Four phyla of fungi produce swimming spores called zoospores. The names for these phyla are Blastocladiomycota, Chytridiomycota, Neocallimastigomycota, and Cryptomycota. Batrachochytrium dendrobatidis is a chytrid associated with the global decline in amphibian populations. Its zoospores infect frogs and other amphibians, producing cysts in their skin that develop into spherical thalli from which the next generation of swimming spores are released. Other chytrids, as well as some of the Blastocladiomycota, produce elongated thalli that release zoospores from sporangia that develop at the tips of branching filaments. Species of Neocallimastigomycota grow in the digestive systems of cows and other herbivores. They are strict anaerobes, poisoned by oxygen, and aid the digestion of plant materials in their hosts. The Cryptomycota, introduced in Chapter 1, have been identified in all kinds of aquatic habitats using metagenomic methods. Some of them infect other fungi and diatoms, but we know very little about these microbes because we have not been able to grow them in culture.
The evolutionary tree of the fungi becomes very complicated when we consider the zoosporic species and it is difficult to be clear about which species fit into which group and whether some should be regarded as fungi at all. Although Olpidium (Figure 7) looks a lot like Batrachochytrium, genetic comparisons show that it is a closer relative of the zygomycetes. So, despite its simple structure and zoospore formation, the classification of Olpidium as a chytrid is questionable. The position of microorganisms called Microsporidia is another problem for taxonomists. Microsporidia are parasites that grow inside the cells of insects, crustaceans, and fish. Some of them cause intestinal infections in people with damaged immune systems. Like many parasites, their genomes have shrunk as they have become more and more reliant upon the activities of their hosts. Microsporidia are related to ‘basal’ fungi, meaning that they seem to be affiliated with the ancestors of the fungi, but it is arguable whether they should be classified as fungi or as a group outside the fungi. This lack of clarity is not surprising because all groups of organisms bleed into one another through their ancestral roots.
Whether or not microsporidia are proper fungi, the study of mycology encompasses a tremendous range of organisms. More than 16,000 kinds of mushroom-forming basidiomycetes have been described and the real number is probably twice as big. For comparison, the catalogue of vertebrate animals is close to complete and zoologists recognize 5,400 mammals and 10,000 birds. Mycologists who are dedicated to the identification of fungi have a lot more to contend with than birdwatchers. A related technical challenge in fungal biology is the problem in defining species. Separate species of birds are characterized by the reproductive compatibility between males and females within the species, their reproductive isolation from other birds, and a suite of visible (morphological) and genetic characteristics. Even in ornithology, however, the spread of genetic variation across several subspecies of a bird and the birth of hybrids between species introduce uncertainty in species descriptions. The challenges are bigger in mycology because many fungi will not reproduce sexually in culture and so many of them look the same. This is the reason that new species descriptions rely so heavily on genetic studies.
In the 20th century it was not unusual for a professional mycologist with expertise in taxonomy to spend an entire career describing the microscopic features of a small group of fungi. Shelves of academic monographs have been published about the species within a single fungal genus. This may seem futile until we consider that a monograph on the ascomycete genus Fusarium serves as a guide to identifying a growing list of species that decompose plant debris in soils, cause destructive plant diseases, produce toxic compounds called mycotoxins, and cause human infections. With growing molecular exploration, a lot of this know-how is being lost as the survey of life reveals ever greater variety at the level of genetics. There is, however, no substitute for looking at fungi in nature to appreciate their spectacular diversity. With the company of an experienced guide, a morning walk in the woods after a period of heavy rainfall can provide an inspiring introduction to the fungi. Thanks to macro photographers who celebrate the beauty of the fungi in online collections of colourful images, the armchair mycologist can also be entertained and informed without getting muddy.
Chapter 3
Fungal genetics and life cycles
Genomes
All of the structures produced by fungi, from simple budding yeast cells to long-lived bracket mushrooms, are encoded in genes. The genome of a fungus is a blueprint for its organization and operation. Describing genes as components of a ‘blueprint’ may seem too simple when we think about something as complicated as a living organism. But there is no other type of stored information that is transmitted from one generation to the next, ensuring that black truffles, for example, will develop from the spores of black truffles. This does not mean that every fungus with black truffle genes will look exactly the same. Black truffles come in many shapes and sizes, ranging from wrinkled nuggets the size of walnuts to giants that weigh more than one kilogram and are a sensation at truffle auctions. Truffles with certain versions of genes may be better at getting big, but most of the differences in size and shape are caused by variations in soil type, temperature, rainfall, and the vitality of the truffle colony. Because truffles are mycorrhizal fungi, their development is also influenced by the health of the hazel and oak trees to which they are connected.
The genome of the Périgord black truffle, whose Latin name is Tuber melanosporum, is huge for a fungus. There are a number of ways to compare genomes of different organisms. If we look at the number of pairs of the letters A, T, G, C (nucleotides) organized in the DNA in the cell, we find
that the truffle genome is ten times larger than the genome of the yeast, Saccharomyces cerevisiae, and four times the size of the genome of the cultivated button mushroom, Agaricus bisporus. This measure of genome size can be a poor indication of the number of functional genes that specify proteins: yeast has 6,000 genes, the button mushroom has 10,000 genes, and there are 7,500 genes in the truffle genome. The enormity of the truffle genome is explained by the incorporation of a great deal of non-coding DNA—DNA that does not code for proteins—whose function is unclear. Much of this is in the form of repetitive DNA sequences, including transposable elements that copy themselves and move around the genome.
The sequencing of a genome is one of the most important steps in ongoing research by biologists to understand the mechanisms that control the development and functions of organisms. The second phase of this exploration is to distinguish genes that encode proteins and determine the nature of the proteins encoded by individual genes. This is the process of genome annotation, which is considered part of the research field called bioinformatics. Investigators have identified genes in the black truffle genome that control the production of the flavours and volatile aromatic compounds that rodents, truffle flies, and human truffle lovers find so seductive. Annotation of the truffle genome has also revealed genes that control carbohydrate metabolism and sexual reproduction. Elsewhere in the genome, waiting to be found, are genes that manage the chemical communication between the fungus and the roots of its plant partner, genes that trigger fruit body formation, and genes that shape the beautifully ornamented spores of the truffle.
Yeast
Genetic research is much more advanced in the study of the ascomycete yeast Saccharomyces cerevisiae. There are many reasons why such spectacular progress has been made in understanding how this microorganism works. The most important of these is the ease with which yeast can be grown in culture. Cultures are started by spreading a tiny quantity of yeast cells on agar using a wire inoculating loop, or by transferring cells to liquid growth medium (broth) using a plastic pipette. When the cultures are incubated at 30oC the yeast cells divide by forming buds every 1–2 hours, so that a single yeast produces a group of sixteen cells in less than eight hours, and more than 4,000 cells in a day. Truffles, and many other fungi, do not lend themselves to this kind of straightforward manipulation in the lab. Truffle growers have to wait for seven to fifteen years after inoculating trees before the first harvest. Anyone committed to experiments on truffles must be extraordinarily patient.
Genetic research on Saccharomyces began in the first half of the 20th century. As techniques improved, yeast was adopted as a favourite experimental organism for studies on molecular genetics, cell biology, and evolution. Yeast became a ‘model’ eukaryote in the same way that the gut bacterium Escherichia coli serves as a model prokaryote. To understand how the genetics of yeast are manipulated in the laboratory we need to look at the process of yeast reproduction. Budding yeast cells come in two strains called a and α. These mating types are akin to sexes, although there is no way to tell them apart from their appearance under the microscope. The difference between a and α cells lies in the production of different chemical attractants, or pheromones, and complementary receptor molecules in their membranes. When cells of the opposite mating type detect each other’s pheromones, their surfaces bulge, make contact, and fuse to produce a larger combined cell that contains two complete sets of chromosomes. This is similar to the fertilization of an egg by a sperm in animal biology. In both cases, two cells with single sets of chromosomes combine to form a single cell with two sets of chromosomes. Cells with a single set of chromosomes are called haploid; cells with two sets are called diploid. Life cycle diagrams are helpful in illustrating how the switch between the haploid and diploid state relates to the development of different organisms (Figure 15).
The diploid yeast cells can bud, but when food becomes limited they engage in a special kind of cell division called meiosis that produces four haploid cells each containing a single set of chromosomes. These haploid cells are called ascospores. They sit inside the diploid cell, which is called the ascus, where they develop a thick cell wall. Yeast ascospores function as survival capsules that allow the fungus to wait for a change in environmental conditions. The group of four ascospores is called a tetrad. Tetrads are interesting from a genetic point of view because each of the new spores contains a unique version of the yeast genome. These exclusive genomes originate from the original fusion of two cells (a × α), followed by the rearrangement of genes during meiosis. The same process of genetic recombination in humans produces the differences between sperm and egg cells that result in the profound differences between children, their siblings, and their parents.
15. Sexual reproduction in baker’s yeast, Saccharomyces cerevisiae.
To determine the function of a particular gene, researchers work with mutant strains of yeast that carry mutant versions of the gene. Natural mutations are caused by ionizing radiation, oxidizing agents produced in cells, and errors during DNA replication. Mutant strains are produced in the laboratory by treating cells with chemical mutagens or by exposing cells to ultraviolet light. Both treatments damage DNA structure, producing mutations in a random fashion throughout the genome. Site-directed mutagenesis offers greater precision by introducing mutant copies of specific genes into yeast chromosomes.
Tetrad analysis is a technique used to determine whether or not a particular strain of yeast is carrying a single mutation. Tetrad analysis is also used to map the positions of genes on the sixteen chromosomes in yeast. Tetrads of ascospores are produced by crossing cells of the two mating types and researchers separate the four ascospores from each tetrad into spots arranged in a grid on a culture plate. Between ten and twenty tetrads are processed in this way on a single culture plate, producing forty to eighty spots occupied by single spores. Ingredients added to the growth medium in the plates are used to select for cells with a particular genetic makeup. The growth patterns that develop when the plates are incubated provide information on the nature of the mutant gene. If, for example, two of the four cells from a tetrad form colonies and two do not grow, this may be evidence that yeast is disabled by the mutant version of the gene. This encourages further experiments to determine how the normal gene operates. The conventional technique for separating spores from a tetrad uses a microscope and glass dissection needles controlled with a device called a micromanipulator. Skilled investigators can separate up to sixty tetrads in an hour, but this is exceedingly tedious work. In recent years, the method has been simplified with the automation of some of the steps. Researchers are also working on a hands-free technique that sorts cells using a flow cytometer that reads a unique genetic barcode introduced into each tetrad.
Another important method in yeast genetics is called two-hybrid screening. This utilizes a reporter gene within an engineered strain of yeast to identify interactions between different proteins encoded in the genome. The study of protein–protein interactions is crucial for understanding the function of different genes and their protein products. The combination of decades of research on yeast and development of increasingly powerful methods in molecular genetics and cell biology has enabled investigators to learn more about the way that yeast works than any other eukaryote. This does not mean, however, that we understand everything about this single-celled fungus. Even though its genome was published in 2001, the function of more than 10 per cent of the genes in Saccharomyces is unknown. Fission yeast, Schizosaccharomyces pombe, is another single-celled ascomycete that has been studied in great detail. Its genome was sequenced in 2002 and encodes almost 5,000 genes. When fission yeast divides, an internal septum grows across the midpoint of the cell producing two identical ‘daughter cells’. This is completely different from budding in Saccharomyces. The molecular control of cell division in fission yeast has significant implications for cancer research.
Filamentous fungi
The genetics of filamentous ascomycetes i
s not understood in as much detail as yeast genetics, but a few species have become important experimental organisms. Neurospora crassa has been studied by geneticists since the 1940s and was used in experiments that linked gene expression to the synthesis of enzymes. The life cycle of Neurospora is more complicated than yeast. Like yeast, there are two mating types. Instead of the direct fusion of growing cells that occurs in yeast, Neurospora uses airborne spores called microconidia to fertilize cells on the surface of developing fruit bodies. The fruit bodies of Neurospora are called perithecia. Fertilization creates cells that contain nuclei of both mating types. Fusion of a pair of these nuclei followed by meiosis produces ascospores. In Neurospora, meiosis is followed by a mitotic division so that groups of eight spores are formed rather than the tetrads of Saccharomyces. These spores are arranged in single file inside tubular asci that extend through a hole in the top of the perithecium and blast them into the air above the fungal colony. The perithecia are less than 0.5 millimetres in width and contain up to 300 explosive asci. (The function of asci as pressurized cannons was introduced in Chapter 2.) The Neurospora genome contains 10,000 genes that encode proteins. The large number of genes relative to yeast reflects the formation of multicellular fruit bodies and the development of the branched colonies of filamentous hyphae during the feeding phase of the life cycle.
Basidiomycete fungi that produce mushrooms engage in cell fusion and meiosis to form spores like the ascomycetes, but there are some fundamental differences in the mating processes in these groups. In mushrooms, the fusion of cells occurs long before nuclear fusion (fertilization), meiosis, and spore formation. Colonies keep growing after merger and delay the processes of recombination until the combined organism has formed a fruit body. The details vary from species to species. Feeding mycelia of the shaggy mane or lawyer’s wig mushroom, Coprinus comatus, are common inhabitants of garden lawns. Hyphae that form these mycelia are divided by septa into compartments that contain a single nucleus. Hyphae of this kind are called homokaryons. When hyphae of compatible strains meet in the soil their cell walls fuse to produce a heterokaryon in which each compartment contains one nucleus from each homokaryon (Figure 16). This condition with two nuclei per compartment is maintained by the formation of tiny branches on the outside of the cell, called clamp connections. Each clamp connection acts as a bridge that allows nuclei to flow between compartments. Some books refer to homokaryons as monokaryons and heterokaryons as dikaryons.