The Fifth Kingdom - Chapter 10

FUNGAL GENETICS
-
MENDELIAN AND MOLECULAR

Hotlinks to:  Nuclear division - marker genes and crossing-over
sexual compatibility - bipolar/tetrapolar - intersterility - parasexuality
extranuclear inheritance - genetics and plant pathology -
recombinant DNA and gene cloning

expression of exogenous genes in yeast - expression of genes in filamentous fungi
molecular taxonomy and population genetics - genome projects - mycoviruses


Introduction

Genetics is the discipline that seeks to understand the ways in which the information needed to reproduce an organism is stored within it, and how that information may change and be reassorted before it is passed on to the next generation. In recent years, we have also become concerned with how this information can be changed in a directed way by human intervention. This chapter attempts to show how fungi are useful tools in some areas of both Mendelian and molecular genetics. If your background in this area is sparse, you will find some useful introductory information in Chapters 1 and 9. If you still have trouble with what follows, I recommend that you consult genetics texts such as 'Principles of Genetics' 7th Edition, by E.J. Gardner and D.P. Snustad, published by John Wiley and Sons, New York, and 'Essential Fungal Genetics' by D. Moore and L. Novak-Frazer, published in 2002 by Springer Verlag, Berlin.

In the simplest terms, genetic information (the genome) is maintained in the cell as long, linear sequences of nucleotide base pairs which make up DNA molecules. The order in which these bases occur constitutes the genetic code, and this code specifies the sequences of amino acids required to build all the proteins necessary for the construction and operation of the living organism. DNA molecules can be very long, incorporating many thousands of base pairs, and are called chromosomes. The genome of prokaryotes (bacteria) is contained in a single, usually circular chromosome found in the cytoplasm. The genome of eukaryotes (organisms with nuclei) is contained in two or more (often many more) chromosomes, which are contained in a nucleus, a special command module separated from the cytoplasm by two membranes.

The eukaryotic plants and animals differ from each other in many ways, but both are basically diploid. This means that their nuclei contain two matched sets of chromosomes: (usually one set originally derived from a male gamete, one set from a female gamete). So each chromosome has a 'double.' Most genes on each chromosome have a counterpart, called an allele, on the 'double.'  This allele affects the same characters, though not necessarily in the same way.

For example, one allele of a particular gene makes pea plants tall, while the other allele makes them dwarf. If a tall plant is crossed with a dwarf plant, there will be more tall offspring than dwarf offspring. Plants will be dwarf only if both alleles are of the dwarfing kind. This shows that one allele can mask another: we say that the 'tall' allele is dominant, the 'dwarf' allele, recessive. This makes genetic analysis difficult, and also makes it hard to breed pure lines of many diploid organisms, because it is almost impossible to eradicate recessive genes, since you can't tell whether they are present or not (though it is easy to pure-breed for recessive colour genes such as those expressed in white rats and mice.)


The vast majority of fungi are haploid, which means that their nuclei contain only a single set of chromosomes. The agaric Coprinus cinereus has a genome size of about 37.5 Mb, organized into 13 chromosomes. The haploid condition gives fungi certain advantages over diploid organisms for genetic studies, since there are no competing alleles, and every gene is potentially capable of being expressed in the phenotype (the physical manifestation or incarnation of the organism). This absence of masking makes genetic analysis much easier. The advantages of using fungi in genetic studies are as follows:

(1) The mycelia of almost all fungi are populated with haploid nuclei (Oomycetes, being chromistan rather than eumycotan, are atypically diploid), and many fungi form large numbers of uninucleate, haploid spores. These can be used to study naturally occurring or induced mutations.

(2) The hyphae of closely related eumycotan fungi can fuse with one another (anastomose) locally during normal assimilative growth, exchanging nuclei and thereby producing heterokaryons (mycelia containing genetically different nuclei).

The heterokaryotic condition confers great flexibility on many conidial fungi, helping them to cope with different substrates and conditions. Heterokaryons can be investigated under controlled conditions by isolating spores or hyphal fragments, and are used by geneticists in the complementation test (see below). The production of heterokaryons may also be an essential step toward a long-delayed sexual fusion, as when basidiomycetes initiate dikaryotization by anastomosis between sexually compatible mycelia.


(3) Hyphal fusions also lead to exchange of cytoplasm, producing heteroplasmons. These make it possible to study extranuclear genetic phenomena, and fungi are particularly valuable for the investigation of cytoplasmic inheritance.

(4) The phenomenon of crossing-over, a vital part of the process of genetic recombination, can be most elegantly studied in ascomycetes like Neurospora or Sordaria. These fungi have very short life cycles, and conveniently arrange the 8 nuclei resulting from meiosis and the subsequent mitosis in a linear sequence within the ascus. One nucleus goes into each ascospore, and the ascospores are arranged in single file within the narrowly cylindrical ascus. The ascospores in this 'ordered tetrad' can be individually cultured and tested in various ways. Using appropriate marker genes:
(a) first-division segregation can be distinguished from second-division segregation;
(b) reciprocal and non-reciprocal chromosomal exchanges can be detected;
(c) chromosomes can be mapped;
(d) interference can be studied. (All the terms just mentioned are discussed in more detail below).

(5) The phenomenon of somatic crossing-over was first seen in the fruit fly, Drosophila, but it can be much more easily studied in fungi. Somatic nuclear fusions occur, with low but predictable frequency in fungal heterokaryons. The resulting diploid nuclei occasionally undergo mitotic crossover. Some of the somatic diploid nuclei which have undergone mitotic cross-over can revert to the haploid condition through irregular forms of mitosis. These haploid nuclei have thus undergone genetic recombination without benefit of sex. The process is called parasexuality. Thanks to their production of large numbers of uninucleate spores expressing specific genetic markers (e.g. colour, or nutritional deficiencies), conidial fungi such as Aspergillus nidulans are especially well suited for investigations of this phenomenon.

(6) Fungi can be handled rather like bacteria -- many pure cultures can be stored in a small space, and the generation time is short -- yet fungi are eukaryotic, so results are much more applicable to the other major kingdoms, animals and plants.

Fungal genetics is not without its difficulties. Fungal nuclei are often very small, and we cannot do the kind of analysis of chromosomal arrangement at the metaphase stage of nuclear division that is possible in many plants and animals.   Here's why.

Nuclear division  These drawings (courtesy Dr. J. Aist) follow a fungal nucleus through a normal mitosis, which takes about 5 minutes.  It is apparent that fungal division is not like that in other organisms.  The spindle develops inside the nuclear envelope. There is no metaphase plate. The chromosomes are very small and not very clearly visualized. Their disjunction is not synchronous. Most of the division happens inside an intact nuclear envelope, which eventually elongates, constricts and finally gives rise to two daughter nuclei. 

It is not possible to count chromosomes as easily as in many other organisms, and we have to rely on features discussed below, such as spore colour and assimilative abilities, to investigate genetic traits.


The small size of conidia, ascospores and basidiospores makes them difficult to handle individually, and the necessity for sterile technique to avoid contamination doesn't make things any easier. But with practice, all these handling difficulties can be overcome. Fungi have been widely used to study recombination and gene action. But they have been little exploited in studies of population genetics. This may be partly because it is often hard to decide what a fungal individual is: it has such a diffuse 'body,' and through anastomosis the mycelia of eumycotan fungi often become heterokaryotic, containing nuclear material from several different genomes. Nevertheless, the potential for such studies remains, and is beginning to be exploited in studies of biological species complexes such as that represented by the binomial 'Armillaria mellea'.

Investigating Crossing-over in a Fungus using Marker Genes

Crossing-over is a normal part of the major process called meiosis. As meiosis begins, the diploid cell has two sets of chromosomes. Each chromosome has already replicated itself, and so is composed of two parallel strands or chromatids. Each chromosome comes to lie parallel to the same (homologous) chromosome from the other set: in the illustration the two `white' chromatids represent one homologous chromosome, and the two `black' ones, the other. If we assume that the `black' chromatids carry a gene for dark-coloured ascospores, and the `white' chromatids carry a different allele of the same gene, one that will produce light-coloured ascospores, then (a) shows what happens in the absence of crossing-over, and (b) shows what transpires when a crossover occurs.

10 crossing over.gif (7034 bytes)

In the simplest crossover, shown in (b), a break occurs at the same place in one of the 'white' chromatids and one of the 'black' chromatids. The ends rejoin, but in a new arrangement: the part of the 'black' chromatid carrying the dark ascospore gene is now joined to part of a 'white' chromatid and vice versa. When the four chromatids separate, they will represent new combinations of genes.

This happens in the real world with ascosopore colour in the dung-inhabiting fungus Sordaria, as you can see in the picture below, when a dark-spored 'wild-type' strain is crossed with a pale-spored mutant.

Ascospore colour.jpg (97791 bytes)

As you can see below, more than one crossover can happen between two homologous chromosomes.

10 double crossover.gif (34356 bytes)

This strange but vitally important process of genetic recombination accounts for the unpredictable mixes of parental genes that occur in the offspring of sexual eukaryotes. Crossing-over ensures that sexually reproducing organisms vary in many ways, and so remain physiologically flexible. Crossing-over is one of the main mechanisms involved in providing the pool of variability on which natural selection acts.

If we have appropriate marker genes, like the ascospore colour gene just mentioned, we can use the incidence of crossing-over to find out roughly where these genes are in relation to the centromere (the point at which the chromatids are functionally joined, and the last thing to separate at mitosis). How can we do this? We begin by assuming that a chromosome is equally likely to break anywhere along its length. If this is true, then the further away from the centromere a marker gene is, the more likely it is to be involved in a crossover. Also, if we have two linked marker genes, the further apart they are on a chromosome, the more likely they are to be separated by a crossover. This kind of information allows us to make chromosome maps showing the relative (though not the absolute) locations of our marker genes.


Our map-making rests on the assumption that we can keep track of the products of meiosis. In most organisms we simply cannot recover and analyze all the nuclei arising from one meiosis. But amazingly enough, we can do it in some ascomycetes, because their meiosis takes place in a long, narrow tube called an ascus. The first illustration (above) shows how the products of the divisions lie in a straight line, so that their exact origin can be traced. The example I gave above involving light and dark coloured ascospores is in fact a real one. In Sordaria fimicola, ascospore colour is determined by a single gene. Wild-type ascospores are dark, but there is a mutant strain with pale spores. Sordaria fimicola is homothallic, but the mating of a normal dark-spored strain with a mutant pale-spored strain derived from it can be used to demonstrate some features of crossing-over. In this particular mating, if no crossover involving the ascospore colour gene has happened, there will be four dark ascospores at one end of the ascus, four light ones at the other end, as in (A). But if the segment of chromosome bearing the colour gene has been crossed-over, then each half of the ascus will contain a pair of light spores and a pair of dark ones, as shown in (B). These pairs can appear in several different sequences, depending on which of the chromatids undergo crossing- over. Crossovers can take place between any two of the homologous chromatids, so there are four possibilities for single crossovers: 1-3, 1-4, 2-3, 2-4.

In fact, crossing-over can be even more complex than I have just described, because it can happen twice between a particular pair of chromatids; or one chromatid can exchange segments with both of its homologues. Some of these possibilities are shown in the second illustration. Of course, we can't watch these events, but we can explain the ascospore arrangements resulting from crosses between strains with two marker genes by diagrams such as that given above. Not all genes express themselves so immediately and unequivocally as that determining ascospore colour, but the process of segregation works just the same for any gene. In order to analyze other kinds of markers which don't express themselves visibly in the ascospore, we have to physically pick out the ascospores (this calls for great dexterity and lots of practice), and grow them individually in culture. The sequence of the spores inside the ascus is recorded, and helps in the interpretation of the subsequent genetic analysis.

As we have already seen, if no crossing-over happens between a particular gene and the centromere, the four ascospores at one end of the ascus will all be of one genotype, and the four at the other end will all be of the other genotype. This arrangement is called the 'first division segregation pattern' because the two versions of the gene separate at first division meiosis. But if crossing-over has happened between the gene and the centromere, the two different versions of the gene are not separated until the second division of meiosis. This arrangement is called a 'second division segregation pattern,' and, as the diagram shows, there can be four such patterns, which occur with about equal frequency (look at the illustration again). Any particular gene will show a definite frequency of crossing-over, which naturally increases as its distance from the centromere increases. The recombination frequency for any gene will equal half of its frequency of crossing-over. This is because only two of the four chromatids are involved in any particular crossover. If we observe a squashed perithecium, and find that of 20 asci, 8 show evidence of crossing-over in the ascospore colour gene, we can say that the frequency of crossing-over for our marker gene is 40%, and the recombination frequency is 20%. That figure is also a useful way of placing the marker gene on a chromosome map. One map unit is arbitrarily defined as the distance between linked genes (genes on the same chromatid) that will give 1% recombination. The gene mentioned above is 20 map units from the centromere. If a second marker gene has a recombination frequency of 30%, this means that it is 10 map units further from the centromere than the first marker. It could be only 10 map units from that first marker, but it could also be 50 map units away, on the other side of the centromere.

With patience and dexterity, a two-factor cross can be done with the ascomycete, Neurospora crassa (Sordariales), using two linked marker genes (with alleles A and a, B and b). Three main ascospore patterns will emerge.
(1) The parental ditype, AB AB AB AB ab ab ab ab: if there is no crossing-over between the two marker genes, the two tetrads of ascospores will reflect the characteristics of the respective parents. (2) The tetratype pattern, e.g. AB AB Ab Ab aB aB ab ab: when a single crossover happens somewhere between the two marker genes, four kinds of ascospore result, two parental types and two recombinants.
(3) The non-parental ditype, Ab Ab Ab Ab aB aB aB aB: if two crossovers occur between the marker genes, all the products will be reciprocal recombinants, arranged in two tetrads. None of the products have the same combination of genes as either of the parents. The relative frequencies of these three patterns can be used to calculate the linkage distance between the two marker genes, and to deduce their positions relative to each other and the centromere.

It can also be used to discover which of the two markers is closer to the centromere, and whether the markers are on the same or opposite sides of the centromere. For example, we analyze the ascospore arrangements resulting from a two-factor cross, and find that there are 56 parental ditype asci, 44 tetratype asci, and 0 non-parental ditype asci. What can we deduce from these data? If the marker genes were unlinked (i.e. not on the same chromosome), the frequency of parental ditype and non-parental ditype asci would be expected to be the same. Since no non-parental ditype asci are recorded, we can assume that the two markers are linked (i.e., on the same chromosome). In order to be able to place the markers in their correct relationship to each other and the centromere, we need to analyze the 44 tetratype asci further. We note that there are three arrangements:

(i) 24 are AB AB Ab Ab aB aB ab ab

(ii) 19 are AB AB ab ab AB AB ab ab

(iii) 1 is AB AB aB aB Ab Ab ab ab

The marker genes could theoretically be arranged in one of three ways with respect to the centromere:

(/) Centromere -- Aa -- Bb

(//) Centromere -- Bb -- Aa

(///) Aa -- Centromere -- Bb

Ascospore pattern (i) above is a result of first-division segregation of the Aa marker, and second-division segregation of the Bb marker (the B and b alleles have been exchanged, the A and a alleles haven't). The crossover that produced this arrangement must have happened between the Bb gene and the centromere, but not between the Aa gene and the centromere. If the two markers are on the same side of the centromere, Aa must be closer to the centromere than is Bb (gene arrangement /). But ascospore pattern (i) could also be explained by gene arrangement (///), in which the marker genes are on opposite sides of the centromere. So far, only gene arrangement (//) can be ruled out. However, if we now look at ascospore pattern (ii), it is clear that both Aa and Bb segregated at second division. If we assume there was only a single crossover, this means that it must have taken place nearer to the centromere than either Aa or Bb, and between the centromere and both markers. So both marker genes must be on the same side of the centromere, and gene arrangement (///) can be excluded.

So, by a process of elimination, we have shown that only one of the three possible gene arrangements, (i) Centromere -- Aa -- Bb, fits all the observed facts. Even the `oddball' ascospore pattern (iii) can be explained by a two-chromatid double crossover, between Aa and the centromere, and between Aa and Bb (you can easily work this out on paper: it is not one of the examples shown in Fig. 10.2, but can be visualized if the uppermost example is revamped with the crossover nearest the centromere happening between Aa and the centromere, rather than between Aa and Bb).

We can now calculate the `map distances' of the marker genes from each other and from the centromere. Of the 100 asci examined, 25 (patterns i + iii) had a crossover between Aa and Bb, while 20 (patterns ii + iii) had a crossover between the centromere and Aa. Applying the appropriate formula (half the number of recombinants, divided by the total asci observed, multiplied by 100), we find that the distance between Aa and Bb is 12.5 map units, and the distance between the centromere and Aa is 10 map units.


Interference occurs when crossing-over at one point reduces the chance of another crossover in nearby regions of the chromosome. This phenomenon is detected by studying crossovers of three or more linked genes. Since the centromere itself acts as a marker, we have essentially a three-gene system in tetrad analysis, which is therefore a good way of studying interference.

The events discussed above involved truly reciprocal crossovers, in which exactly equivalent segments of chromatids are exchanged. But sometimes the exchange is not exactly equal. This is called non-reciprocal recombination, or gene conversion, and if very closely linked marker genes are studied, it is found that crossovers are actually more often non-reciprocal than reciprocal. This phenomenon is explained by the breakdown or excision of short lengths of DNA during recombination, and their replacement by replication from another chromatid. Once again, fungi like Neurospora have been very useful in elucidating gene conversion.

Mutant genes can act as markers enabling us to investigate the genetics of fungi. The kinds of mutant genes available affect such features as morphology, colour, mating type, and nutritional requirements. In some morphological mutants, the growth rate or branching pattern of hyphae is altered, with various effects on colony morphology. Neurospora crassa has 'button' and 'ropy' mutants. Other morphological mutations affect reproductive structures: Aspergillus nidulans has 'stunted' conidiophore, and 'bristle,' in which the conidiophore has no conidium-producing apparatus at its apex. Colour mutants usually affect spore colour: Aspergillus niger has 'white,' 'fawn' and 'olive' mutants.


Biochemical mutants are perhaps the most useful markers. Biochemical mutants usually require some nutrient that is not needed by the wild type. Such mutants are called auxotrophs. A minimal medium is concocted for the wild type (for Neurospora crassa this contains only inorganic salts, including a nitrogen source, sucrose, biotin and agar). Samples of the fungus are exposed to a mutagenic agent such as ultraviolet light, then plated out on the minimal medium and also on a complete medium, which contains malt extract and yeast extract in addition to the ingredients listed for the minimal medium. If a strain is found that will grow on complete medium, but not on minimal medium, some biochemical deficiency is suspected. Now a little detective work is called for. This strain must be systematically tested to see what it needs, by attempting to grow it on minimal medium with additions of mixed vitamins, or mixed amino acids, or nucleic acids. If the minimal medium plus mixed vitamins keep it alive, then it is grown on minimal medium supplemented with individual vitamins. In this way the specific requirement of the auxotroph can be pinpointed.

Fermentation mutants arise spontaneously in yeasts, resulting in inability to ferment a particular sugar. Resistance mutants also arise spontaneously in wild populations, but their frequency of occurrence increases if the organisms are exposed to antibiotics, antimetabolites or other deleterious influences: such mutants are actively sought in the laboratory. The fungus is grown in a concentration of the deleterious substance high enough to inhibit normal growth: resistant mutants are the only ones to survive. Suppressor mutants overcome or compensate for any deficiency induced by an earlier mutation, and cause an apparent reversion to the wild-type. Physiological mutants apparently change the biochemistry of the fungus subtly, altering its reactions to some environmental influence, such as temperature or light. One mutant of the zygomycete, Phycomyces blakesleeanus, has normal morphology, but its sporangiophores no longer grow toward the light.

Another group of biochemical mutants are those which produce greater than normal amounts of particular substances. Although this kind of mutant hasn't been subjected to very much genetic analysis, it is sometimes economically important. The commercially exploited strains of Penicillium chrysogenum that produce such large amounts of penicillin are mutants of this kind.


One Sex, Two Sexes, Many Sexes

Sexual reproduction will introduce more genetic variation to a population if the genomes which meet, and are then reassorted during meiosis, come from different individuals. That statement may sound strange and even superfluous to you, since you belong to a species in which such behaviour is not only natural, but obligatory. But in many cases, an individual fungal mycelium can and does keep its sexuality to itself -- its hyphae can produce sex organs of both kinds which go through the processes of sexual fusion and produce a viable zygote. This condition is described as homothallism. Homothallic taxa are very useful if we simply want to demonstrate sexual behaviour in fungi, since we don't have to worry about providing a suitable mate. The advantage of this system in Nature is probably two-fold: to permit sexual reproduction when no appropriate compatible mycelium can be found (the lonely spore hypothesis), and to perpetuate particularly successful genotypes, which would tend to be reassorted, and therefore diluted, by outbreeding.

Many fungi, however, have evolved some form of reproductive differentiation of individual mycelia: we call this phenomenon heterothallism, and it enforces outbreeding. One approach is sexual dimorphism: the production of two kinds of sexual structure which look and act differently, and are often developed on different mycelia. In some fungi, both kinds of sex organ can be formed by a single mycelium, but only gametes originating from different mycelia can fuse. This implies genetic control of sexual reproduction through the development of mating types that incorporate incompatibility genes. These make sex impossible between strains of the same mating type. In many fungi, mycelia may be morphologically indistinguishable, yet invisible incompatibility factors can prevent their mating. Incompatibility can prevent anastomosis, or prevent karyogamy. In fungi like the ascomycetes, where fusion of assimilative hyphae does not initiate the sexual process, vegetative incompatibility is not a barrier to sexual reproduction, and is often determined by entirely separate genes, so that a single species may be divided up into a number of vegetative compatibility groups (VCGs). Such ascomycetous taxa as Cryphonectria parasitica (Diaporthales), Neurospora crassa (Sordariales), and Fusarium moniliforme (anamorphic Hypocreales), contain many VCGs. In the basidiomycetes, where fusion of ordinary, undifferentiated assimilative hyphae is a prerequisite to the establishment of the dikaryophase, and dikaryotization a prerequisite of karyogamy, vegetative incompatibility can effectively prevent sexual reproduction.


Basically, heterothallism implies that a haploid nucleus can complete the life cycle only if it mates with another haploid nucleus carrying a different mating-type factor. Heterothallism is the fungal equivalent of the separate sexes found in many plants and animals. As we shall see, the fungi, despite their restricted genome and relatively consistent organization, have evolved many and complex variations on this sexual theme.

The simplest kind of genetic system that can ensure outbreeding consists of two different alleles, which we can label 'A' and 'a,' at the same locus. Pairs of mycelia carrying the same allele will be incompatible (A with A, or a with a), while pairs of mycelia with different alleles (A and a) will be compatible. This system effectively divides a population into two categories, and has the same effect as division into two sexes. This two-allele system is found in all groups of fungi other than the most highly evolved basidiomycetes. Examples are the zygomycetes, Rhizopus and Phycomyces, species of the ascomycete genera Neurospora, Ascobolus and Sclerotinia, and species of the teliomycete genera Puccinia and Ustilago.

Bipolar and Tetrapolar Mating systems

In Aphyllophorales, Agaricales and gasteromycetous basidiomycetes, compatibility is determined by one or two genes, but each of these may have many different alleles. Only two or four alleles are present in any given dikaryon, at a single locus or at two loci. If all compatibility alleles occur interchangeably at one locus, the mating system of the fungus is called bipolar; if they are found at two loci, the mating system is called tetrapolar. If the alleles occur at a single locus, the offspring of a single basidioma will be of two different mating types. If the alleles are at two loci, offspring of a single basidioma will be of four mating types. Although the products of meiosis in the basidiomycetes are not an ordered tetrad, as they are in the cylindrical asci of some ascomycetes, it is still possible to culture the four basidiospores arising from an individual meiosis, and use them in compatibility trials.

In bipolar fungi (most smuts, some gasteromycetes, Coprinus comatus), the single locus at which all compatibility alleles occur can be called A. Now we can label the alleles in a given dikaryon: A1 A2 (they must be different, or the dikaryon wouldn't form in the first place). Other dikaryons will probably have different alleles, which we can call A3 A4, A5 A6, etc. Random matings in populations with such diverse mating-type alleles can be almost 100% successful. Remember that random matings in populations of 2-allele organisms would be only 50% successful. Now we can see why a multiple-allele system may be more desirable than a two-allele system.

In tetrapolar fungi (most Aphyllophorales, Agaricales and gasteromycetes), we can label the two loci A and B. For a dikaryon to be fertile, the alleles at each of the loci must be different -- we can label them A1 B1 A2 B2. The haploid (monokaryotic) mycelia derived from this dikaryon will be of four kinds: A1 B1, A2 B2, A1 B2, and A2 B1. You can easily work out that only 25% of random matings among these siblings will be successful. Of course, matings of non-sibling monokaryons will again work much better: if we match up the four genotypes just listed with monokaryons derived from a dikaryon that is A3 B3 A4 B4, success should be complete. But what if the allele at one of the loci is the same as in our original strain, so that its alleles can be listed as A1 B3 A4 B4? What should be the percentage success of matings between this and the products of this and the original strain (A1 B1 A2 B2)? Work it out on paper. Your answer (which should be 50%) represents the number of fertile dikaryons that will result. But if you did this experiment, you would probably find that you finished up with many more dikaryons than you expected. This is because dikaryons can form between partially incompatible monokaryons, though such dikaryons will not be able to produce fruit bodies. In the example I just gave, there could be as many as 87.5% dikaryons (37.5% of them sterile), and only 12.5% total incompatibility.

It has been found that the genes at the two loci often control different parts of the dikaryotization process. In the basidiomycetes, Coprinus lagopus and Schizophyllum commune, clamp connections develop only if the dikaryon is heterozygous (has different alleles) for the A locus. For example, A1 B1 A2 B1 would have hyphae with clamps, A1 B1 A1 B2 would not. Nuclear migration is controlled by the B locus, and would fail in A1 B1 A2 B1.

Of 230 species of Aphyllophorales, Agaricales and gasteromycetes examined, 10-15% were homothallic, about 35% were bipolar heterothallic, and about 55% tetrapolar heterothallic. It has been estimated that Schizophyllum commune probably has about 340 + 120 different A alleles, and 64 + 12 different B alleles. Estimates in some other basidiomycetes are of the order of 100 different alleles for each locus, though the bird's-nest fungi, Cyathus striatus and Crucibulum vulgare, are believed to have only about 10 alleles for each locus.

Secondary homothallism can occur in heterothallic fungi. If an ascus contains only four spores, as in Neurospora tetrasperma, instead of eight, there can be a compatible pair of nuclei in each spore. Similarly, if a basidium bears only two spores, as in Agaricus brunnescens, each of these may also contain two compatible nuclei. Homothallism is possible, even in species with four-spored basidia. If an extra mitosis happens in the basidia, two compatible nuclei may find their way into some of the basidiospores.

Homothallism can also be introduced in what would otherwise be a heterothallic fungus by mating-type switching. In addition to the functional mating type allele at the active locus, Saccharomyces cerevisiae has 'silent' copies of mating-type alleles at two other loci. A site-specific endonuclease cuts the double stranded DNA at the active locus. The resulting gap is then repaired by splicing in DNA from one of the loci at which the silent copies reside. This often means that one allele is replaced by the other, so the mating-type of the organism is switched. Similar switching occurs in another yeast, Schizosaccharomyces pombe, and in the filamentous ascomycetes, Sclerotinia trifoliorum, Chromocrea spinulosa, and Glomerella cingulata, though the mechanism is still obscure in those fungi. The switching in Chromocrea and Sclerotinia happens in only one direction. If the mechanisms involved are like that found in Saccharomyces, it is likely that only one of the mating-type alleles is present in a silent form. We do not yet know how much fungal homothallism can be accounted for by mating-type switching. In some fungi, self-sterile spores with a single nucleus, and self-fertile spores with two nuclei, are both developed in the same fruit body. This kind of mating behaviour is called amphithallism.

Recognizing the existence of compatibility genes is one thing, understanding how they work is another. The best-documented compatibility system is that of the yeast, Saccharomyces cerevisiae. Here there is a single locus with two alleles. Each mating type secretes a constitutive polypeptide pheromone which causes cells of the opposite mating type to become arrested in the G1 stage of the cell cycle. Such arrested cells agglutinate and undergo plasmogamy and karyogamy. If the resultant diploid cells are starved, they will undergo meiosis and produce haploid meiospores. Each stage of this process is apparently under the control of mating-type genes. These genes are regulated by DNA binding proteins encoded by the mating-type alleles. One of the alleles contains a unique sequence of 747 base pairs, and encodes two regulatory polypeptides. The other allele has a unique sequence of 642 base pairs, and encodes two polypeptides, of which only one is known to be regulatory. The mating-type genes of other fungi are currently being isolated and characterized, and we should soon know how representative S. cerevisiae really is. It will be a tremendous challenge to explain how the hundreds of separate alleles we know to exist in some individual basidiomycete taxa differ, and are regulated.


Intersterility

Compatible mating-types are not always enough to ensure successful sex. Sometimes, mating fails despite apparent compatibility. There is therefore another genetic system, which we can call an intersterility system, that can override the usual incompatibility system. Unfortunately, we don't know nearly as much about the basis of this system as we do about incompatibility. The kinds of barriers involved are either prezygotic, preventing fertilization, or postzygotic, resulting in hybrids of reduced fertility or meiotic offspring less fit than the parents.

Prezygotic barriers exist between closely related populations of many well-known basidiomycetes, including Armillaria, Collybia, Coprinus, Laccaria, Paxillus, Pleurotus, Ganoderma and Heterobasidion. Since intersterility is usually complete, particularly in sympatric populations, the intersterile groups are equivalent to biological species. In some of these fungi, DNA reassociation or DNA restriction fragment patterns have shown that the intersterile groups are also genomically divergent. Sometimes two entirely intersterile sympatric populations are partly interfertile with a third population from another area. We do not yet know whether this third population could act as bridge between the other two solitudes. In Ustilago cynodontis, intersterile populations and partly interfertile 'bridging' strains coexist within what appears to be a single complex species.

Postzygotic barriers are present when mating occurs, but most of the resulting spores are not viable. In closely related heterothallic Neurospora species, the reproductive barriers appear to be mostly postzygotic. Intraspecific crosses yield viable ascospores, but interspecific crosses produce largely non-viable ascospores.


Parasexuality

Ascomycetes and basidiomycetes can be easily distinguished when they reproduce sexually. In this phase (the teleomorph) they form characteristic fruiting bodies (ascomata and basidiomata) bearing unique meiosporangia (asci and basidia) from which, as we have seen, the products of a single meiotic event can be isolated and analyzed. Many of these fungi reproduce asexually as well, producing what are called anamorphs, which form mitospores called conidia, and often occur well separated in time and space from the teleomorph. In fact, we know thousands of anamorphs which have not yet been persuaded to metamorphose into a teleomorph. Many of these go on, generation after generation, in the asexual condition, and it now appears highly probable that many of them have entirely lost the ability to produce a teleomorph, thus becoming anamorphic holomorphs.

We know that one of the most vital functions performed by the teleomorph is genetic recombination. This reassortment of the gene pool during meiosis broadens the ability of the population to cope with the stresses imposed by changing environments. Conidial fungi, which are often highly opportunistic, and grow on a wide range of substrates, might seem to be especially in need of the flexibility conferred by genetic recombination. One of their responses to this perceived need for genetic diversification is to become heterokaryotic: to acquire more than one kind of nucleus as a result of one or more anastomoses. But we now know that they have also evolved a special mechanism for generating some genetic recombination without sex. We call this process, parasexuality. The parasexual cycle has four stages.

(1) Fusion (anastomosis) of adjacent somatic hyphae, and exchange of nuclei, establishing a heterokaryon.
(2) Fusion of different nuclei in the vegetative hyphae, to form somatic diploids.
(3) Somatic recombination (mitotic crossing-over).
(4) non-meiotic reduction of the altered nuclei via aneuploidy (loss of individual chromosomes) to the haploid condition.

This sequence of events is rare, happening in fewer than one conidium in a million, but the number of conidia produced by most conidial anamorphs is astronomical, so parasexuality is a practical means for producing genetic variation. We don't yet know how widespread this phenomenon is among the conidial fungi, but it has been detected in species of Aspergillus, Acremonium, Fusarium and Verticillium, and is probably common.

It is worthwhile to compare sexuality and parasexuality.
(1) Sexual reproduction is a highly organized, often precisely timed process, which is genetically programmed. Parasexuality involves a sequence of uncommon events which seems to operate by chance, rather than by design.
(2) In sexual reproduction, nuclear fusion is often mediated by genetic factors, expressed as 'mating types,' happens in highly specific structures, and often involves many pairs of compatible nuclei. In the parasexual cycle, nuclear fusion is an isolated event, not mediated by mating-type factors, not found in specialized structures, and involving only individual nuclei.
(3) During meiosis, crossing-over probably takes place in every homologous pair of chromosomes, and multiple crossovers are common. During somatic recombination, crossing-over commonly involves only one or a few chromosomes, and never happens as often as during meiosis.
(4) In meiosis, segregation happens in a highly organized way during two specialized nuclear divisions. Somatic haploidization probably occurs as a result of successive chromosome losses from an aneuploid nucleus (2n-1) over several mitotic divisions until the stable haploid is reached.

The factors that initiate sexual reproduction vary enormously from one fungus to the next, presumably because of their diverse ecological adaptations, so it is very difficult to make generalizations, though special media have been concocted to persuade such important genetic tools as Neurospora to undergo sexual reproduction on demand. The parasexual cycle can be encouraged in various ways. Camphor vapour selects for somatic diploids in some fungi. In species with uninucleate conidia, the best approach is to produce a heterokaryon between two auxotrophic mutants (each of which has a different biochemical deficiency), then grow its conidia on minimal medium. Neither of the original auxotrophs will be able to grow, but diploid conidia will grow, since the chromosomes from one parent compensate for the deficiency in the other set, and vice versa (this is called complementation, and the diploid is described as being prototrophic). This technique has been used in Verticillium albo-atrum, Aspergillus niger, and Aspergillus nidulans, and yields about one diploid conidium in 106-107 conidia. The subsequent frequency of mitotic recombination can be increased by X-rays, U/V, mitomycin and nitrous acid. Finally, to complete the cycle, low concentrations of p-fluorophenylalanine or benlate (Benomyl) stimulate haploidization.

We can see the potential advantages of the parasexual cycle to an asexual fungus, but is it of any use to the geneticist? As it happens, it can be used to determine linkage groups, the order of genes, and the position of the centromere. The genetic recombination achieved is on a much smaller scale than in meiosis: only one or two chromosomes are involved, and the possibility of multiple crossovers is so low that it can be ignored. This means that linkage analysis is much easier. The original diploids are heterozygous for the various marker genes. Those in which crossing-over subsequently occurs will become homozygous for any marker genes that are distal to the point of crossover. The relative frequencies with which such markers become homozygous is an indication of their relative distances from the centromere.


Extranuclear Inheritance

Some genetic phenomena can't be explained by reference to nuclear or chromosomal events. The logical corollary of this is that the determinants may be transmitted in cytoplasm rather than in nuclei. In some heterothallic fungi, the volume of cytoplasm that accompanies one of the nuclei during a sexual fusion may be much greater than that associated with the other nucleus. Alternatively, if one side of the fusion involves a microconidium or a spermatium, this must inevitably bring much less cytoplasm to the union than does the receiving partner. This sometimes results in the offspring resembling the parent that contributed more cytoplasm, and implies the existence of cytoplasmic genes. It has been shown that in Aspergillus glaucus, attributes such as spore germination, growth rate, pigmentation, and density of cleistothecia, are under cytoplasmic control. A well-known example of cytoplasmic control is the 'poky' mutant of Neurospora crassa. This grows more slowly than the wild-type, and cannot be speeded up by dietary supplements. If 'poky' is crossed with the wild-type, the poky condition is transmitted only when the 'poky' strain forms the perithecium initial, which means that it is essentially the maternal parent.

Another well-known example of extranuclear inheritance is the petite strain of Saccharomyces cerevisiae, which arises with a frequency of about 1 cell in 500. Such cells give rise to smaller than normal colonies, which can respire only anaerobically, even when oxygen is present. This deficiency is due to the absence of important respiratory enzymes such as cytochrome oxidase and succinic dehydrogenase. The mitochondria are defective. Whole colonies can be converted to 'petite' cells by growing them on medium containing 3 ppm acriflavine. Diploid petite cells don't reproduce sexually, but diploid hybrids derived from petite and normal haploid cells respire aerobically and can form ascus-like meiosporangia. If the meiospores are cultured, all are normal, and petite cells arise among their offspring only in the ordinary 1:500 ratio. Normal yeast cells contain cytoplasmic genes (in mitochondria) controlling the synthesis of respiratory enzymes. Petites arise by mutations in the mitochondrial DNA. When a cell containing this cytoplasmic mutant fuses with a normal haploid cell, the extranuclear genes from the normal cell render the resulting diploid normal again, and normal mitochondria find their way into any resultant haploid cells, which will all therefore be normal.


Genetics and Plant Pathology

Plant breeders try to produce, not only higher-yielding varieties of crop and garden plants, but also new disease-resistant strains. This is done by finding natural defence mechanisms that are present in wild relatives of the economically important host plant. Painstakingly, the plant breeders introduce the resistance genes to the crop plants. Although such new cultivars may be immune to a particular fungal disease for a few years, eventually a new race of the fungal pathogen appears which can overcome the resistance of the plant. Analysis of this endlessly repetitive cycle of resistance and susceptibility led to the theory of the gene-for-gene relationship between host and pathogen. This suggests that the evolutionary paths of host and pathogen have been so closely linked for so long, that for every gene in the host that is capable of mutating to give resistance, there is a corresponding gene in the pathogen which can mutate to overcome that resistance.

Passalora fulva, a hyphomycete, (formerly called Fulvia fulva and Cladosporium fulvum)causes leaf mould of tomato. Three genes that confer resistance to this fungus are known, and tomato varieties exist which carry none, one, two, or all three of these genes. With the aid of these host varieties, eight races of Passalora fulva can be discriminated. The most efficient way to differentiate these races is with three tomato varieties which have, respectively, resistance genes 1, 2 and 3, as can be seen from the Table. If you examine the eight columns which give the responses of the three tomato varieties to the different fungal races, you will see that each column differs from all the others. This means that any of the eight races can be identified by testing it against only three tomato varieties.


Interactions of races of Passalora fulva with 3 tomato varieties

Tomato with resistance gene

Passalora races

0

1

2

3

1 + 2

1 + 3

2 + 3

1 + 2 + 3
       1 R S R R S S R S
       2 R R S R S R S S
       3 R R R S R S S S

That is how prevalence and spread of many important plant pathogenic fungi is monitored. It is also the mechanism by which the existence of new physiologic races of pathogens is discovered. The more genes for resistance we recognize, the more pathogenic races can be distinguished. Almost 200 races of the flax rust fungus, Melampsora lini, have been identified by their reactions with 18 host varieties. Puccinia graminis subsp. tritici, which causes wheat rust, has well over 200 races, and the number is growing steadily in response to the efforts of the plant breeders.

The genetics of resistance have also been explored in Venturia inaequalis, the apple scab fungus, which is a bitunicate ascomycete. It was found that the genes controlling virulence exist in virulent and avirulent alleles, which segregate regularly in the ascus. Seven of these genes were discovered, and seven apple varieties were found that would enable their presence to be recognized. For example, the avirulent allele of gene 1 didn't affect MacIntosh apples (which might simply mean that MacIntosh carried a corresponding gene for resistance to that allele). Yellow Transparent apple was resistant, not only to avirulent 1, but also to the avirulent alleles of genes 3 and 4. Each of the seven apple varieties had a different resistance gene or genes, which could be identified by exposing the host to various races of V. inaequalis.

The natural testing ground for resistance of potatoes to the late blight fungus, Phytophthora infestans (Oomycota) is central Mexico, where both sexes of the fungus are present, and new physiologic races can arise more readily than elsewhere. Working in this environment, potato breeders have found it more useful to aim for 'field resistance,' which is mediated by many genes with small individual effects, rather than concentrating on a few major resistance genes with all-or-none effects. The war goes on.


Recombinant DNA and Gene Cloning in Fungi

Since fungi have not been among the most important contributors to our knowledge of DNA and how it works, I will not burden you with the usual spiel on DNA, its functions and its replication: you can get that from any first year Biology text book. In addition, for an overview of recombinant DNA technology, you should refer to a recent text on gene cloning. However, although fungal DNA is essentially the same as that of animals and plants, it is present in relatively much smaller quantities.  Here are some representative genome sizes:  Mycoplasma 0.6 million base pairs, Haemophilus 1.8 mbp, yeast (Saccharomyces) 14 mbp, Drosophila 180 mbp, Human 3,100 mbp, Onion (Allium) 18,000 mbp, fern (Ophioglossum) 160,000 mbp, amoeba 670,000 mbp.

Compare that with the number of genes in various genomes: Haemophilus 1,700; Escherichia coli 4,000; yeast (Saccharomyces) 6,000; Drosophila 14,000; Arabidopsis (mustard) 27,000; Human 35,000.

The techniques of molecular biology have not only given us a great deal of detailed information about the genetic material, and even the actual sequence of base pairs which make up parts of the genomic DNA, but also permit the movement of genetic material from one organism to another, and the expression of certain genes from one organism in another. I will set the scene by outlining the processes involved in moving genes from one organism to another. Recombinant DNA technology usually involves the following steps.
(1) Cells of a host (often the bacterium Escherichia coli) are broken, and their DNA extracted.
(2) This DNA includes plasmids, small closed rings of extrachromosomal DNA, which may be used as vectors for the introduction of foreign DNA. The vectors are separated from the other DNA by ultracentrifugation.
(3) Special enzymes called restriction endonucleases cleave the plasmid vectors, and leave them as linear sequences of DNA with 'sticky' ends (unpaired bases).
(4) DNA from the donor organism (the source of the desired gene) is isolated and then treated with the same restriction endonucleases, producing additional linear sequences with sticky ends that match those of the cleaved plasmids.
(5) Vector DNA and donor DNA are mixed: sticky ends rejoin, by complementary base pairing, in various configurations -- vector ends re-join, vector joins donor, donor joins donor. In a few cases the desired joinings happen, producing a closed loop, which is part vector DNA, part donor DNA. 
(6) The sugar-phosphate backbone of the DNA is then properly repaired by an enzyme called a DNA ligase.
(7) The modified vectors are mixed with E. coli made permeable by treatment with a calcium salt. This allows some of the bacteria to pick up modified vectors, and so be transformed.
(8) The transformed cells that bear the desired donor gene can now be isolated, with the help of selectable marker genes previously incorporated in the vector, and can subsequently be propagated on a large scale.

Why are yeasts and filamentous fungi now being used in gene cloning, if bacteria are such suitable hosts? Fungi are valuable because:
(1) Many of the donor genes we want to clone are eukaryotic. Bacteria aren't ideal hosts for this job, because their mechanisms of gene transcription and translation are so different from those in eukaryotes. In fact, even if suitably modified plasmids are successfully introduced to Escherichia coli, relatively few eukaryotic genes will be expressed by this prokaryote, because it does not know how to deal with introns (it doesn't have any). This is obviously not a problem in the fungi, which are all eukaryotes. The yeast, Saccharomyces cerevisiae, bypasses some of the roadblocks encountered when E. coli was the principal host available. S. cerevisiae can, for example, glycosylate proteins, fold them, or carry out other post-translational modifications which must be made if some eukaryotic proteins are to become functional.
(2) Saccharomyces cerevisiae and such filamentous fungi as Aspergillus nidulans are genetically well-explored, and useful mutations are available in many of their biochemical pathways. 
(3) Yeasts can be grown and handled in very much the same way as the bacterium, Escherichia coli: simplicity itself compared to the tissue culturing of animal cells.

Two techniques are commonly used to transform yeast cells; one requires the removal of the cell walls, the other uses entire cells. The first technique is carried out as follows.
(1) The yeast cells are treated with ß-mercaptoethanol, a reducing agent, which facilitates subsequent digestion of the cell wall.
(2) The wall is digested by mixtures of glucanases derived from snails or bacteria.
(3) The resulting 'protoplasts (often called spheroplasts) are washed and suspended in a stabilizing solution (0.6 M KCl or 0.8 M sorbitol) to which is added the foreign DNA (vector incorporating the desired sequences).
(4) Uptake of the plasmid DNA during protoplast fusion is promoted by adding polyethylene glycol.
(5) The transformed protoplasts are then allowed to regenerate a wall, and are grown on a selective medium (one containing a specific antibiotic, or with a particular food substrate, depending on which marker genes were used) which will allow only appropriately transformed cells to grow (because only they carry the appropriate marker gene, which came with the vector, and confers resistance to that antibiotic or the ability to metabolize that particular substrate).


The alkali salt method permits transformation of intact cells. Cells are incubated in lithium acetate to make them competent, i.e. receptive to exogenous DNA. The DNA is then incorporated in the presence of polyetheylene glycol 4000. Although transformation is less efficient than with protoplasts, the procedure is simple and quick, cells can be stored for weeks without loss of competence, and the problem of diploid formation during protoplast fusion is avoided.

The first demonstration that yeast could be transformed with exogenous DNA was made in 1978, using a recombinant bacterial plasmid carrying the Saccharomyces cerevisiae gene for an enzyme needed in the synthesis of leucine (LEU 2). This gene had earlier been recognized in E. coli because it complemented a mutation in the bacterium that had caused the loss of the same enzyme. Several other yeast genes have now been cloned in E. coli by complementation of other bacterial mutants. These are useful markers which can be incorporated in the exogenous DNA along with the desired gene: their uptake and subsequent expression in yeast cells allows recognition and selection of yeast cells which have been appropriately transformed; that is, which now carry the desired donor gene.

Most strains of Saccharomyces cerevisiae contain up to a hundred '2µm plasmids' per cell. Each plasmid has about 6300 base pairs. Hybrid plasmids made up of the entire 2µm sequence, plus the LEU 2 yeast gene, plus a bacterial vector sequence, efficiently transform yeast cells that lack the LEU 2 gene (As a consequence of the bacterial vector sequence DNA having been replicated in a bacterium, the 2µm plasmid also works in E. coli, so it can serve as a 'shuttle vector'). The complementation of the LEU 2 gene means that those cells which have been properly transformed can be selectively isolated on leucine-free medium, and subsequently multiplied. It has also been demonstrated that the hybrid plasmid replicates in transformed cells. However, it appears that smaller plasmids containing only a fragment of 2µm DNA are more versatile, giving higher frequencies of transformation, and more copies of the plasmid in each transformed cell (up to 300). All stages of gene-cloning can be carried out in yeast, but it is usually more efficient to amplify recombinant plasmids in E. coli. The most important aim of the cloning exercise may be to obtain gene products, but cloning also lets us produce a lot of homogeneous DNA, which can then be used in the sequencing of genes.

Expression of exogenous genes in yeast

Yeast genes generally have the following components: (1) upstream promoter elements which include constitutive or regulated promoters (positive or negative); (2) 20-400 bp downstream, a TATA promoter element (so named because it incorporates the base sequence, thymine-adenine-thymine-adenine); (3) 30-90 bp downstream, a transcription initiating site which initiates production of mRNA; (4) protein-coding sequences; (5) transcription termination signals. Transcription of the inserted DNA depends on the presence of a promoter sequence that is recognized by the host RNA polymerase.

10_fungal_gene.gif (6831 bytes)


Highly expressed yeast genes such as alcohol dehydrogenase I (ADH1) or glyceraldehyde-3- phosphate dehydrogenase (G3PDH) usually have very high mRNA levels, so most methods of expressing exogenous genes in yeast have concentrated on the production of high mRNA levels. This involves using multiple copy plasmids to boost the number of gene sequences per cell, and fusing coding sequences to efficient yeast promoters to increase transcription.

Yeast genes may contain both constitutive and regulated promoters, which serve to initiate transcription. Where both are present, the constitutive sequences are active at all times during cell growth, and may produce a base level of gene expression which can be modified by other upstream sequences. Regulated promoters need to be activated before they will initiate transcription, but will in some cases produce much higher levels of gene expression. Researchers initially tended to use constitutive promoters with high mRNA levels, especially those from the genes mentioned above (ADH1 and G3PDH), but found that while these gave high yields of homologous proteins, they produced much lower amounts of heterologous (donor) proteins. If large amounts of the desired protein are deleterious to the host cell, it is better to use regulated promoters, which are not derepressed until required. Good examples are those involved in galactose metabolism, such as GAL1, GAL7 and GAL10, which are repressed by glucose and derepressed by the addition of galactose to the medium. If cultures are grown with glucose or glycerol as carbon source, these promoters will remain repressed until almost all the glucose has been metabolized. If glucose is absent, and galactose is added to the medium, these promoters can be induced about 1,000-fold. Galactose induction is controlled by the GAL4 and GAL80 proteins. The GAL4 protein is a positive promoter that binds to specific DNA sequences upstream of the coding sequences of genes regulated by galactose. The GAL80 protein is a negative regulator which binds to the GAL4 protein, preventing it from activating transcription. If galactose is added, it binds to the GAL80 protein, stopping this from interfering with the GAL4 protein, which can then go about its business of promoting transcription. Recently, hybrid promoters have been developed, combining strong constitutive promoters with upstream sequences of regulated genes. One of these has been used in the controlled expression of human interferon


Brewer's yeast must be provided with sugars if it is to produce alcohol. If yeast could be transformed so that it possessed an amylase (a starch-degrading enzyme), production of ethanol would be simpler and cheaper. Various amylase genes from bacteria, yeasts and filamentous fungi have now been cloned and expressed in Saccharomyces cerevisiae: commercial exploitation of these should soon be possible.

The cellulose and hemicellulose in wood represent almost limitless potential substrates for the fermentation industry. Complete degradation of cellulose to glucose requires the activities of three successive enzymes: endoglucanase, exocellobiohydrolase and -glucosidase. Trichoderma reesei secretes all three cellulolytic enzymes, but normal cultures of the fungus aren't used to produce these enzymes commercially, because all three enzymes are inhibited by their end-products. The strategy has been to isolate mutant, highly cellulolytic strains of Trichoderma reesei, then isolate the genes for the three enzymes and place them in vectors with control sequences appropriate for their expression in a suitable host. In 1987 the T. reesei gene for endoglucanase was cloned, characterized and expressed in S. cerevisiae. Endoglucanase and exoglucanase from the bacterium, Cellulomonas fimi, have been cloned, expressed and secreted in yeast in a regulated manner by attaching their coding sequences to the melibiase promoter and signal sequences from Saccharomyces carlsbergensis. Although yields are still too low for commercial exploitation, there is optimism that higher-yielding strains will be developed, and that cellulolytic brewer's yeast will be able to clarify beer, and provide cheaper fuel alcohol.

It appears that recipient yeast strains can take up and maintain exogenous DNA even without the mediation of vectors. The brewing industry has achieved this in two ways. Beer normally contains dextrins that are not degraded by brewing yeasts. These dextrins give a beer greater body, and a higher caloric content. The light beers which are so popular today (for reasons that escape me) have had these dextrins removed by an exogenous enzyme, glucoamylase. This enzyme is produced naturally by some non-brewing yeasts (for example, Saccharomyces diastaticus). Brewers have therefore tried to get the ability to make this enzyme into their brewing strains, so that these could produce a light beer without assistance. One approach has been to incubate the protoplasts of the brewing yeast with partially purified high molecular weight DNA from the donor yeast. A second approach has been to fuse entire protoplasts of the two yeasts. Unfortunately for this second method, the S. diastaticus brought with it, not only the glucoamylase, but also 4-vinyl guaiacol, which ruined the flavour of the beer. Classical hybridization techniques were then used to segregate the glucoamylase gene from the 4-vinyl guaiacol gene.

The flavour of Brazilian wines is often spoiled by an excess of 1-malic acid. Fusion of the wine yeast protoplasts with those of Schizosaccharomyces pombe, which metabolizes 1-malic acid, produced a hybrid that successfully reduced 1-malic acid levels in the wine. Protoplast fusion has some potential, because some characters important in baking, brewing and distilling are polygenic (controlled by many different genes), or are not well understood genetically. Such characters aren't suitable for enhancement by gene cloning or transformation. In addition, protoplast fusion combines whole genomes, and it is known that increases in ploidy may increase productivity. Intergeneric fusions are often unstable, but if one 'petite' parent (which has non-functional mitochondria) is used, more stable hybrids are produced, probably because the hybrid contains functional mitochondria from only one parent.

Expression of Eukaryotic Genes in Filamentous Fungi

Although for several years yeasts were the hosts favoured by gene-cloners seeking to express heterologous eukaryotic genes, they cannot secrete enzymes in the quantities produced by bacteria. But mycelial conidial fungi such as Aspergillus niger can secrete enzymes more efficiently than either yeasts or bacteria, and are therefore becoming the hosts of choice for expression and secretion of many enzymes, antibiotics and even mammalian pharmaceutical proteins. Transformation in filamentous fungi was first reported for Neurospora crassa in 1979, and transformation systems have now been developed for many other filamentous fungi.

Genes in filamentous fungi are composed of a promoter, a translation initiation region, DNA encoding a secretory signal peptide (where necessary), DNA encoding for the gene product, and DNA sequences for terminating transcription and for polyadenylation.

10 fungal gene.gif (6831 bytes)


When a gene is to be moved to a new host, it is first assembled in a vector plasmid which can replicate in E. coli. A selectable marker may also be incorporated in the plasmid. As mentioned earlier, markers may be selectable because they compensate for some auxotrophic deficiency in the host, or (preferably, since in many cases we are not dealing with auxotrophic hosts) because they confer upon the host resistance to an antibiotic, or the ability to metabolize a particular food substrate. The hyphae of the host fungus are enzymically stripped of their walls, and the transformation of the resultant protoplasts proceeds much as it does in yeasts. Following transformation, the protoplasts convalesce on a regenerative medium which lets them reconstitute their hyphal walls. In fungal transformations, the vector DNA usually becomes integrated into the host genome. Bovine chymosin, a mammalian protease used in cheese-making, has been successfully expressed and secreted in Aspergillus nidulans. The cDNA sequence encoding the chymosin was attached to transcriptional, translational and secretory control elements of the glucoamylase gene from Aspergillus niger. All four units were incorporated into a suitable vector, which was used to transform Aspergillus nidulans. Active human interferon 2 and a bacterial endoglucanase have also been cloned in A. nidulans, this time using promoters from the A. niger glucoamylase gene and the A. nidulans alcohol dehydrogenase gene. Human tissue plasminogen activator (tPA, a protease used to dissolve blood clots) has been expressed and secreted in A. nidulans. One of the vectors constructed for this purpose is illustrated here.

10 plasmid vector.gif (7471 bytes)


Molecular Taxonomy and Population Genetics

Fungal identification is often difficult, even with good, mature specimens of the usual reproductive structures in hand. If all we have are sterile mycelia, or fungus-inhabited substrates, identification has been virtually impossible. But now, with the advent of a variety of molecular techniques, the impossible just takes a little longer.

We can detect the activity of specific enzymes. We can use probes to identify particular base sequences in the DNA. We can do immunoassays, using antibodies raised in a mammal against unique components of the organism. Or we can use sodium dodecylsulphate polyacrylamide gel electrophoresis (more succinctly known as SDS-PAGE). This technique separates the proteins in any mixture by their molecular weight. An electrical field is used to draw the protein molecules through a porous gel. Smaller molecules move more quickly and so travel further in a given time. Eventually, the concentrations of the various protein molecules are made visible by staining with dyes or silver-based reagents, and the resulting spatial and intensity pattern of bands compared, often by computer, with those derived from known organisms. References describing these techniques are given at the end of the chapter.

 

 


It has become possible to compare parts of the DNA or RNA sequences in the genome of different organisms with a view to establishing their degrees of biological relatedness. Rhizopogon is a 'false' truffle, a hypogeous, mycorrhizal basidiomycete with a closed basidioma, a convoluted hymenium, and non-shooting basidia. For anatomical reasons, this fungus has been thought to be related (although nobody knew how closely) to 'normal' epigeous members of the Boletaceae, which produce basidiomata with a stipe, a cap, hymenium-lined tubes, and spore-shooting basidia. In recent molecular studies of these fungi, a number of fragments of Suillus mitochondrial DNA (mtDNA) were cloned and hybridized with mtDNA from other members of the Boletaceae. This showed that

                                                    PCR

Using the Polymerase Chain Reaction (PCR) technique, researchers can now routinely replicate very small samples of DNA thousands of times, and ultimately produce enough DNA to permit analysis of its base pairs.

This technique showed that 15 different regions of the mitochondrial genome of Rhizopogon subcaerulescens are virtually identical to those of fourteen species of the 'normal' bolete genus, Suillus. This was surprising, because not only does the order of these 15 regions differ among species of Suillus, but Rhizopogon and Suillus have traditionally been placed in different families or even different orders. Their molecular similarity, at least as far as this has been explored, is in striking contrast to their morphological divergence.

In defence of classical taxonomy, I must point out that many mycologists have long believed that Rhizopogon is a secondarily reduced or sequestrate (non-spore-shooting) derivative of the genus Suillus. It is encouraging that this relationship has now been dramatically affirmed. This work also demonstrates either that major morphological changes may not be reflected by corresponding changes in the genome, or that we have not been looking in the right places to find the genetic reflection of those differences. It also emphasizes that our concepts of fungal relationships must be based on as many kinds of information as possible (not just morphological, and not just molecular).

Armillaria mellea, perhaps the only truly diploid basidiomycete, produces assimilative mycelial clones up to 400 metres in diameter, and up to several hundred years old, infecting many trees, and several different clones may be present. When homokaryons anastomose, nuclei migrate but mitochondria do not, so the resulting mycelium is uniform in its nuclear component, but has at least two sectors with different mitochondrial genotypes. Therefore, an examination of mitochondrial DNA polymorphisms can now help us discover the history of those clones.

The uses of molecular techniques in mycology are multiplying, and I must warn my readers that it is almost impossible for any publication to keep pace with the latest developments in this field. However, the attempt must be made!

DNA sequencing, usually after amplification by PCR (the polymerase chain reaction), is now being used to identify important fungi, such as commercially grown species or serious plant pathogens, or to find the appropriate taxonomic niche for fungi whose taxonomic position is problematic. A small region of the genome of several individuals in one species is compared to the DNA sequence of the equivalent region in other taxa.. Currently the region being sequenced and compared most commonly is the rRNA (ribosomal RNA) operon. This operon consists of three genes under the control of a single promoter. The genes are (1) the small subunit gene (SSU), (2) the 5.8S gene, and (3) the large subunit gene (LSU).

Fortunately, in most fungi these genes are arranged in that order, and so can be located and compared. The first gene transcribed is the SSU. After this comes an Intergenic Spacer Region (IGS) which contains an internal transcribed spacer region (ITS 1), the 5.8S gene, and a second transcribed spacer region (ITS region 2). At the other end of the IGS is the LSU.

The rRNA operon is part of a multi-gene family consisting of repeated arrays of operons. Different genes and regions in the rRNA operon have different degrees of sequence conservation (the likelihood that changes will enter the sequence of base pairs over time). The varying pressure for sequence conservation is due to the differing degrees of importance of the different sequences: areas that are less important in the function of the genes tend to vary more than areas that are crucial. This is because any changes in those crucial areas might wreck the gene’s ability to make its product.

The rather mysterious Internal Transcribed Spacer regions (ITS), since they do not themselves code for a gene product, are far more variable than the genes. The ITS regions have proved most suitable for comparisons between related species. The 5.8S gene is small, highly conserved and so of little value for phylogenetic comparison. The SSU is also conserved, but since it is larger than the 5.8S gene, more variation has crept in, and it has been used for comparisons among genera and higher taxa. The LSU is the least conserved of the three genes and has allowed comparisons within and between species.

DNA sequencing after PCR amplification has been used to identify species of Armillaria (and many other fungi). But these techniques are not restricted to identification of known species. They can also be used to connect or compare previously unidentifiable mycelial (non-sporulating) cultures with fungi that have been identified using their sporulating structures (whether sexual [teleomorphic] or asexual [anamorphic]). The DNA profiles of non-fruiting cultures have been compared with the profiles from DNA isolated from fruit bodies of Armillaria, and with this evidence in hand, new species of Armillaria have been described. This raises some problems, such as how the new taxa could be identified without resort to expensive and time-consuming molecular techniques, but the day of a hand-held sequencer surely cannot lie more than a decade in the future!

One of the most recent developments in this field is the establishment of comprehensive diagnostic tests for mixed cultures or other material of fungi, from a range of species of Pythium to a variety of unknown fungi in soil samples.  This is done using species-specific oligonucleotides.  In one case the ribosomal ITS regions for all living cultures of Pythium were sequenced, and a unique oligonucleotide devised for each of them.  These oligonucleotides were then synthesized and dotted in an array on a nylon membrane.  Hybridization to this DNA array was used to identify species present, using sample probes that had been amplified and labelled.  Once this technique has been fully worked out, identification of mixtures of unknown fungi should become possible without need for detailed mycological knowledge on the part of the operator.

Usually, sequencing every fungal isolate in order to identify it is far too time consuming and expensive. Therefore, a quicker and simpler technique based on the polymerase chain reaction (PCR) and the specificity of restriction enzymes (Restriction Fragment Length Polymorphisms or RFLPs) has been devised. This is the "PCR/RFLP" technique. It involves the amplification of a specific gene or region of the genome by PCR. The amplified fragment of DNA is then cut with one or more restriction enzymes, and the resulting restriction fragments are separated using gel electrophoresis (the smaller fragments - those with fewer base pairs - travelling further along the gel) which produces specific banding patterns called profiles. These banding patterns look rather like the bar codes used by check-out scanners to identify products in many stores. The band profiles generated in this way are chosen so that one (or at least just a few) types of profiles are specific to one species of fungus. In this way fungal species can be 'fingerprinted' and compared.

The specificity of the PCR reaction also offers possibilities for identification. DNA-based primers can be identified which have a sequence that is unique - found only in a particular species. Thus when PCR amplification using these primers works, we can say that the fungal isolate being examined belongs to a given species or group of fungi. Such techniques also lend themselves to fluorescent quantification. Commercial techniques using fluorescent quantification have not yet been developed for fungi but are already being used for a number of bacterial pathogens of humans.

In the years ahead we can look forward to molecular help with some of our more difficult taxonomic problems, although I do not foresee a day when our taxonomic concepts are based entirely on DNA sequencing. It is important to stress that a fungus is much more than the sum of its base pairs! Despite this stricture of mine, papers are now being published in which different names are given to fungi that are morphologically indistinguishable - the taxonomic decision is based almost entirely on DNA sequences. It will be interesting to see if such names stand the test of time.

How would you like to be told that two groups of organisms, distinguishable only by their DNA, are being described as different species? Looking at DNA sequence data derived from species we know to be distinct,  we are getting an idea, at least in some groups, of how different fungi must be before they can be considered as distinct taxa.

It might be suggested that if two groups have occupied the same kind of niche, this may have caused them to retain morphological similarities, but they may also have been genetically isolated from each other for so long that the base sequence of their DNA has changed. However, we have to remember that at the end of the day the whole purpose of taxonomy is to help us to identify organisms, and if it becomes too difficult to do this using morphology, can we justify using the more expensive techniques? The answer may be that we need 'functional classification' and phylogenetic classification. The Botanical Code of Nomenclature requires a diagnosis of a new taxon which states how it differs from existing taxa.  It is not unusual for such diagnoses to be largely focused on DNA sequences.

I and many other mycologists think that molecular data is important and exciting, but should be correlated with morphological comparisons and with comprehensive population and mating type studies. This is the ideal we should strive for.

                                    Pyrosequencing

Nowadays, new DNA sequencing techniques, originally developed for genomic studies (see below) are revolutionizing studies of molecular ecology. So-called environmental metagenomics, using techniques such as 454 pyrosequencing, generate literally millions of DNA sequences (often ITS barcodes), allowing thousands of species of fungi to be detected and tentatively identified from complex substrates such as soil. Although the computer analyses involved in these experiments are complex, these powerful techniques are rapidly replacing traditional studies involving culturing and identification of cultures by morphology. The reliability of the results depends on the reliability of the reference databases used to identify the sequences, making the development of DNA barcode databases particularly critical.

Genome Projects

Most people are aware that a large number of laboratories collaborated in the sequencing of the entire human genome.   This was a huge project, yet was completed in 2001, in an amazingly short period of time.  But the entire genomes of many other organisms have now been sequenced.  These include viruses, bacteria, fungi, and several well-known experimental animals and plants.  Saccharomyces cerevisiae (baker's and brewer's yeast) was the first fungus to be sequenced. That took many years and involved 600 scientists (only 16 of whom got their name on the publication). Other organisms sequenced early include the fruit fly, Drosophila melanogaster, the nematode, Coenorhabditis elegans, and the plant, Arabidopsis thaliana.  The genome of Saccharomyces was found to contain about 14 million base pairs with about 6,000 recognizable genes, divided among 16 chromosomes.  The Saccharomyces genome was the first fungal genome to be completely sequenced, but other genome projects have been constructed for such mainstays of genetics as Neurospora crassa, and for a number of pathogens such as Cryptococcus neoformans.  All of these projects will make it easier to place any sequenced fragment, and thus to know where specific genes are, and what their functions are.  The future of fungal genomics looks exciting.

 Over 70 fungi have now been fully sequenced. More and more organisms are being sequenced as it becomes quicker and less expensive to do this. If you have $15,000 to spare for materials, you can get raw data, produced in a few weeks. But it would still take about $400,000 to complete the job. 

The 'genome sequences' or genetic maps for the fungi Aspergillus fumigatus, Aspergillus nidulans and Aspergillus oryzae have now been decoded. Although these three species are from the same genus, they have been found to be as genetically different as fish and man (groups which diverged 450 million years ago), since they share only about 68% of the same proteins. They also differ considerably in genome size, with that of Aspergillus oryzae being 31% bigger than that of Aspergillus fumigatus and 24% bigger than that of Aspergillus nidulans. Intriguingly, over 30% of the 9,500-14,000 genes identified are new to science and of unknown function. Aspergillus fumigatus was identified as a cause of infection as long ago as 1848 and is now the leading infectious cause of death in vulnerable leukaemia and bone marrow transplant patients. Aspergillus nidulans has been a leading experimental genetic system for the last 50 years, whilst Aspergillus oryzae has been used in the Far East for 2,000 years to produce sake (rice wine), miso (soybean paste) and shoyu (soy sauce) (see Chapter 19).

Now we want to know, not just the sequence of bases in the DNA, but also the entire proteome - all the proteins encoded by the multifarious genes.  Finally we need to understand the transcriptome, the ways in which various genes are turned on and off during development in a hierarchical manner.  There appears to be a cascade effect as a small number of genes turn on other genes, and these in turn deploy others (and turn them off again when necessary).  Only then will we know how life really works.

Genomics

Genomics concerns itself with the ways in which genes and other factors interact during development. It used to be assumed that one gene meant one protein, and that the fixed and orderly process of assembly and interaction of proteins would explain all aspects of development.

It has been found that humans and mice both have about 3 billion base pairs, and very comparable numbers of genes (about 25,000), with a very high level of equivalences (it is rare to find a human gene that does not have a mouse equivalent – not more than 1% of the total), so the differences between these two organisms are largely explainable by three processes: changes that have gradually evolved in many of the genes, in the timing of the activation of the genes, and in the duration of that activation (they may be switched on and off repeatedly). It is not so long ago that Hox genes were discovered. They are genes which govern the activity of other genes by enhancing or repressing their expression.

                                        Epigenetics

Epigenetics, an even more recent genetic discovery, involves a similar enhancement or repression of gene action, but without the involvement of other controlling genes. Probably the best understood example of such modifications is DNA methylation, which regulates gene expression without changing the base sequence.  This chemical change may persist through multiple cell generations, and even into succeeding generations of the organism, and in some ways mimics the Lamarckian concept of inheritance of acquired characteristics.

Epigenetics is thus the study of changes in gene expression caused by modifications to ways in which parts of the genome are expressed, not by changes in the base sequence of the DNA itself.  These changes may not be fully heritable, but they do allow certain factors to override the normal expression of genes, causing changes in morphology or differentiation.

In recent years it has been discovered that epigenetic processes can have important effects on the development of an organism.  Most of these changes have been observed and explained in animals, but similar processes occur in fungi, and epigenetics may play an important part in the evolution and development of these organisms.

High Throughput sequencing analysis
in molecular fungal ecology

High-throughput sequencing analysis methods allow us to make detailed analysis of fungal communities, using large sets of samples, and provide ecological information that is far beyond anything previously possible. As more and more named fungal sequences become available in repositories such as GenBank, more sequences recovered can be linked to known species. The procedures do, however, require careful attention to protocols, and it is vital that users are aware of potential problems. Several world leaders in fungal community molecular ecology have come together to produce a valuable and pragmatic “user’s guide” (Lindahl et al. 2013).

Researchers visited 26 pine forests all across North America and collected a total of 600 10-cm-deep soil cores. The samples were quickly preserved, and the fungal DNA in them was extracted and isolated. The researchers then sequenced unique stretches of the DNA that allowed them to identify all of the fungal species present in each sample.

This revealed more than 10,000 species of fungi in the 600 samples, which the researchers then analyzed to determine biodiversity, distribution, and function by geographical location and soil depth. Interestingly, there was very little overlap in the fungal species from different regions - East Coast fungi were not found on the West Coast or Midwest, and vice versa. But although ‘regional endemism’ was strong, similar suites of enzymes showed that they performed the same saprobic and symbiotic roles in all locations (Talbot et al. 2014). This paper by Talbot et al is a very fine example of the way in which molecular techniques can throw light on major ecological problems.  It has excellent illustrations, and I strongly recommend that you all read it.

                                               Proteomics

It is apparent that the very large number of proteins required for the complex activities of a cell, and the extremely complex ways they are folded and configured in order to do what is needed, are not simply coded into the DNA as a series of sequences, since the functional DNA is not enough to do all this without further manipulations, and different proteins may be required at different times. In fact proteins are constructed by the carefully timed operations of small parts of the genome, controlled and coordinated by master genes - turned on and off as required. So now it is not enough to know the base sequence of the DNA, but we also need to know much more about the control; mechanisms and their master genes. Wikipedia puts it succinctly:   "The proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes."  Proteomics was 'invented' in 1997, and is now a growing part of genetic investigations.

                                      Genetic Barcodes

DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA to identify it as belonging to a particular species.  This is not an attempt to identify unknown taxa, but simply to compare a short, known segment of bases with equally short segments in other known organisms, which is believed to establish conspecificity. It is obviously much easier and quicker to use such small and precisely chosen sequences than to compare whole genomes or large segments of them.

 (1) For animals and many other eukaryotes, the mitochondrial CO1 gene: a 648-bp segment of mitochondrial cytochrome c oxidase 1 (CO1) is the standard barcode region. As of 2009, databases of CO1 sequences included at least 620,000 specimens from over 58,000 species of animals.
(2) For land plants, it is the concatenation of the rbcL and matK chloroplast genes.
(3) For fungi,
cytochrome c oxidase 1 (CO1), although not yet widely tested, was found to be suitable in fungi, including many Penicillium spp. (Ascomycota). Because introns were present in some taxa, the barcode region potentially ranges from 642 bp in Hypocrea jecorina to 12.3 kb in Podospora anserina (both Ascomycota).

                      A proposed DNA barcode for Fungi

                In 2012, agreement was reached (see Schoch et al. – more than 150 collaborators) on the barcode to be used for Fungi. An evaluation of several DNA regions - the mitochondrial cytochrome c oxidase subunit 1, three regions from the nuclear ribosomal RNA cistron, regions of three representative protein coding genes (RPB1, RPB2 and MCM7), nuclear ribosomal small subunit (SSU), and nuclear ribosomal large subunit (LSU) - concluded that the internal transcribed spacer (ITS) region was the most appropriate, having the highest probability of successful identification of the regions within the ribosomal cistron across the broadest range of fungi. This region most often provided a barcode gap between the levels of within-species and between-species sequence variation, so was formally adopted, although supplementary barcodes may be needed for some taxonomic groups.

                                 Mycoviruses

Hypovirulence or reduced virulence associated with the presence of dsRNA has become a well recognized phenomenon in some fungal pathogens. The first of these mycoviruses to be well characterized was the so-called 'hypovirus,' so named because it causes hypovirulence (reduced pathogenicity) in the chestnut blight fungus, Cryphonectria parasitica. This virus apparently has no coat protein, and can’t exist outside the fungal cell. It is transmitted from one strain of the fungus to the next during anastomosis (the fusion of somatic hyphae). Fungal mycelia are capable of anastomosis only when they belong to the same vegetative compatibility group (VCG). Diverse fungal populations with many different VCGs will tend to inhibit the spread of the virus. So, while mycoviruses are potentially potent biocontrol agents for fungal pathogens, they are likely to be more effective in pathogen populations with low genetic diversity.

A growing number of mycoviruses have been found in fungi. Some, like the Cryphonectria parasitica hypovirus are associated with symptoms such as reduced virulence of the pathogen (similar viruses have been found in other ascomycetes such as Ophiostoma ulmi and Diaporthe ambigua). Other mycoviruses are cryptic. The best known of the more cryptic mycoviruses are found in the well studied yeast, Saccharomyces cerevisiae. These viruses are associated with the killer yeast phenomenon, where one of the viral genomes codes only for a protein toxin. The other genomes code only for a coat protein and an RNA-dependent RNA polymerase. Neither of these last two genomes have any apparent negative effect on their host. Most mycoviruses do not appear to be closely related, and a number are based on DNA sequences rather similar to those of some plant viruses.

                                            Afterword - Where now?

The implications and applications of current genetic techniques as applied to a wide range of organisms are explored in a fine book by John C. Avise (see reference below). Since it was published in 2003, things will have moved on significantly by now, but it is an excellent way into the maze...
 

 
        Go to Chapter 11
             Go to Table of Contents

© Mycologue publications 2015


Further Reading on Fungal Genetics

Avise, John C. (2003) The Hope, Hype and Reality of Genetic Engineering.  Oxford Univ. Press, New York.

Beggs, J.D. (1981) Gene cloning in yeast. pp. 175-203 (in) Genetic Engineering 2. (ed.) R. Williamson. Academic Press, New York.

Bennett, J.W. and L.L. Lasure (Eds.) (1985) Gene Manipulations in Fungi. Academic Press, Orlando.

Bennett, J.W. and L.L. Lasure (1991) More Gene Manipulations in Fungi. Academic Press, New York.

Berka, R.M. and C.C. Barnett (1989) The development of gene expression systems for filamentous fungi . Biotechnology Advances 7: 127-154.

Blum, H., H. Beier and H.J. Gross (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels.  Electrophoresis 8: 93-99.

Brasier, C.M. (1987) The dynamics of fungal speciation (in) Evolutionary Biology of the Fungi. (Eds) A.D.M. Rayner, C.M. Brasier and D. Moore.   Cambridge University Press, Cambridge.

Bruns, T.D., T.J. White and J.W. Taylor (1991) Fungal molecular systematics. Ann. Rev. Ecol. Syst. 22: 525-564.

Bruns, T.D., R. Fogel, T.J. White and J.D. Palmer (1989) Accelerated evolution of a false-truffle from a mushroom ancestor.  Nature 339: 140-142.

Bruns, T.D. and J.D. Palmer (1989) Evolution of mushroom mitochondrial DNA: Suillus and related genera.  Journal of Molecular Evolution 28: 349-362.

Burnie, J.P., R.C. Matthews, I. Clark and L.J.R. Milne (1989) Immunoblot fingerprinting of Aspergillus fumigatus.  Journal of Immunological Methods 118: 179-186.

Glass N.L. and G. Donaldson (1995) Development of primer sets designed for use with PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol.  61: 1323-1330

Harrington, T.C. and D.M. Rizzo. 1999. Defining species in the fungi. pp 43-71 (in) Structure and Dynamics of Fungal Populations. (ed.) J.J. Worrall.  Kluwer Academic Press.

Harrington, T.C. and B.D.Wingfield (1995) A PCR-based identification method for species of Armillaria.  Mycologia  87: 280-288.

Herskowitz, I. (1988) Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiological Reviews 52: 536-553.

Hibbett, D. S., Y. Fukumasa-Nakai, A. Tsuneda and M.J. Donoghue (1995) Phylogenetic diversity in shiitake inferred from nuclear ribosomal DNA sequences.  Mycologia   87: 618-638.

Jackman, P.J.H. (1985) Characterisation of microorganisms by electrophoretic protein patterns (in) New Methods for the Detection and Characterisation of Micro-organisms. (ed.) C.S. Gutteridge. Wiley, U.K.

Jahnke, K-D., G. Bahnweg and J.J. Worrall (1987) Species delimitation in the Armillaria mellea complex by analysis of nuclear and mitochondrial DNAs.  Trans. Brit. Mycol. Soc. 88: 572-575.

Lindahl BD, Nilsson RH, Tedersoo L, Abarenkov K, Carlsen T, Kjřller R, Kőljalg U, Pennanen T, Rosendahl S, Stenlid J, Kauserud H (2013) Fungal community analysis by high-throughput sequencing of amplified markers – a user’s guide. New Phytol 199: 288–299.

Martin, C.E. and S. Scheinbach (1989) Expression of proteins encoded by foreign genes in Saccharomyces cerevisiae.  Biotechnology Advances 7: 155-185.

Moore, D. and L. Novak-Frazer (2002) Essential Fungal Genetics Springer, Berlin 357 pp.

O'Donnell K. and E. Cigelnik (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous.  Molec. Phylogenetics Evol: 103-116.

O'Donnell K, E. Cigelnik and H. Nirenberg (1998)  Molecular systematics and phylogeography of the Gibberella fujikuroi species complex.   Mycologia 90: 465-493.

Old, R.W. and S.B. Primrose (1985) Principles of Gene Manipulation: an Introduction to Genetic Engineering. 3rd Edn. Blackwell Scientific, Oxford.

Perkins, D.D. (1987) Mating-type switching in filamentous ascomycetes. Genetics 115: 215-216.

Scheinbach, S. (1983) Protoplast fusion as a means of producing new industrial yeast strains. Biotechnology Advances 1: 289-300.

Strathern, J.N., E.W. Jones, and J.R. Broach (Eds.) (1981) The Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance. Cold Spring Harbor Monograph Series. Vol. 11A. Cold Spring Harbor, New York.

Strathern, J.N., E.W. Jones, and J.R. Broach (Eds.) (1982) The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression. Cold Spring Harbor Monograph Series. Vol. 11B. Cold Spring Harbor, New York.

Talbot JM, Bruns TD, Taylor JW, Smith DP, Branco S, Glassman SI , Erlandson S, Vilgalys R, Liao H-L, Smith ME, Peay KG (2014) Endemism and functional convergence across the North American soil mycobiome  Proc Natl Acad Sci USA 111: 6341-6346.

Taylor, J.W. (1986) Fungal evolutionary biology and the mitochondrial DNA. Experimental Mycology 10: 259-269.

Van Brunt, J. (1986) Fungi: the perfect hosts?  Biotechnology 4: 1057-1062.

Vellinga, E.C. (2008) Fungal genomes. Mycena News (Mycol. Soc. of San Francisco)

Volk, T.J., H.H. Burdsall and M.T. Banik (1996) Armillaria nabsnona, a new species from western North America.   Mycologia 88: 484-91.

Watson, J.D. and A Berry (2003) DNA The secret of life. A.A Knopf, New York. 446 pp.