1. MODUL ADVANCED MICROBIOLOGY SOURCE OF VARIATION by Gayuh Rahayu Microbiology Study Program Department of Biology Faculty of Mathematic and Natural Sciences Bogor Agricultural University 2010

2. Introduction Genetic variation in the fungal life cycle permitting genetic flexibility to overcome the its relationship with host and adapt to environment. The mechanism by which the fungi introduce genetic variation is influenced by the reproductive process. In fungi, the primary source of genetic variation is reproductive process, but genetic variation may also result from mutation.Reproductive system in fungi can be divided into three groups. i.e. asexual, sexual and parasexual reproduction. The asexual methods of reproduction commonly found in fungi can be summarized as: 1) fragmentation of soma, each fragment (arthrospore) growing into a new individual, 2) fission of somatic cells into daughter cells, 3) budding of somatic cells or spores, each bud producing a new individual, and 4) production of mitotic spores, each spores germinating to form a germ tube that grows into a mycelium. Sexual reproduction gives a higher opportunity to produce genetic variation, followed by parasexual and asexual. A major feature of sexual reproduction is that haploid nuclei undergo karyogami to form a diploid nucleus. Karyogami may occur after plasmogamy. Meiosis occurs resulting in four haploid nuclei. Genetic variability of the progeny result because of 1) genetically different cells are brought together to initiate the sexual cycle and 2) crossing over and random assortment occur during meiosis. New gene combination arisen as a result of sexual reproduction may contribute to the overall fitness of the individual. In certain fungi, a dikaryotic (N+N) phase often occurs in short or longer time depending on the fungus, prior to diplophase.The plasmogamy, karyogamy and meiosis in sexual reproduction have to occur in specialized cells and in successive mode. Differing from sexual reproduction, the parasexual cycle is a sequence involving 1) heterokaryon formation, 2) diploidization and 3) haploidization. Parasexual cycle occurs in anamorphic fungi. The main difference between sexual (meiosis) and parasexual cycle (mitotic) is the restoration of haploid cell from diploid cell. The process of each step in the parasexual cycle is independent of one another and the frequency of occurrence is very low. However, parasexual become a major recombination type for asexual fungus. Sexual Compatibility On the basis of sex, most fungi may be classified into three categories: a) hermaphroditic (monoecious) in which thallus bears both female and male organs that may or may not be compatible.

3. b) dioecious. Some thalli may only bear female or male sex organs. Very few fungi have been discovered to be dioecious. c) sexually undifferentiated in which sexually functional structures are indistinguishable as male or female. The vast majority of fungi fall into this category. Whatever the sexuality of the fungi, the strain should undergo plasmogami, prior to karyogami and meiosis. Plasmogami in fungi may occur through several ways i.e. gametangiogami, somatogami and gametogami. 1) Gametangiogami or gametangial copulation. Gametangiogami means fussion of two gametangia. The gametangia can be devided on the bases of its role, i.e male and female gametangium. Saprolegniales produced antheridia and oogonia and some ascomycetes such as Pyronema domesticum produced antheridia and ascogonia. These gametangia can occur in the same specialized hyphal branch. Self fertilization can occur in these hermaphroditic fungi if plasmogami occurs between neighbouring gametangia. Gametangiogami accomplished by the transfer of the entire protoplast of one gametangium into another. 2) Somatogami.Members of Basidiomycetes lack sexual organs can be a homothallic. A single basidiospore germinates, giving a mycelium. Homotahallism may bring this mycelium to be able to form basidiocarp through somatogami, eventhough no genetic difference in the nuclei involved. 3) Gametogami or planogametic copulation. Depending on the type of gamet (zoospores), the copulation can be divided into three categories, i.e. fusion of isogametes (gametes morphologically similar but physiologically different), fusion of anisogametes (morphologically different usually in size) and fertilization of non-motile female gametes (egg) by a motile gamete. In some species gamet produced from one gamtegangium will not fuse. Gamets that fertilize eggs usually produced in the anteridium. Fusion of planogametes takes places in water to form motile zygotes. Genetic Control of Sexuality Sexual reproduction involves the union of gametes or gametangia, bringing together their nuclei to form dikaryon and subsequently the zygote. The sexual organ can be either differentiated (gametangiogamy) or undifferentiated (somatogamy). There is another characteristic of sexual reproduction in fungi has to be considered, that is the existence of mechanisms that regulate sexuality. This means there is a control process whether the fungus is able to mate with a compatible partner. The concept of sexuality can be differentiated into homothallism, heterothallism and secondary homotahllism.. This sexuality is controlled by intranuclear gene called mating type gene. Homothallism, Homothallism is characterized by inbreed mating (self fertilization). A homothallic fungus can complete its sexual reproductive cycle

4. within or upon a thallus derived from a single uninucleate spore. In all of homothallic fungi, the single nucleus contains all genetic requirements for sexual expression. So the key of concept of homothallism is that karyogamy and meiosis can occur without involving a second mating partner. If mating needs a second partner, there is no requirement that the nuclei represent different mating type. The majority of all fungi are probably homothallic. Homothallism is predominates in the zoosporic fungi and ascomycetes, and also occurs in a few basidiomyctes. Eventhough homothallism seems not to create genetic variation, variation may occur due to mutation. However the mutation might not be in the mating type gene. As a result of these potential mutation and recombination, genetic variation in homothallic fungi is low. Heterotahallism.These fungi are self sterile and do not carry all genetic requirements for sexual reproduction in a single nucleus. Mating between two compatible homokaryotic cells or thalli is required to produce a zygote having a full complement of genetic requirements. This phenomenon is called heterokaryosis. Heterotahllism is often controlled by sexual dimorphism in few fungi. Heterotallism is controlled by incompatibility mechanisms. Secondaryly homothallism. In some fungi, spores may contain two nuclei of different mating type. Germlings arising from these spores are therefore self- fertile and behave as homothallic eventhough in reality they are heterothallic. Heterokaryon formation (heterokaryosis) provides a great deal of genetic variation in the fungi. Phenotypically, the mycelium with heterokaryon may different from its parents, since it contains different cytoplasm and nuclei. Heterokaryosis may occur in a fungal thallus in four ways (Fig 1): 1) by the germination of heterokaryotic spores, which will give rise to heterokaryotic soma. 2) by the introduction of genetically different nuclei into a homokaryon. 3) by mutation in multinucleate, homokaryotic structure and multiplication and spread of mutant nuclei among the wild type nuclei. 4) by fusion of two haploid nuclei in the homokaryon to form a diploid nucleus (diploidization) and multiplication and spread of diploid nucleus among the haploid nuclei. Both mutation and diploidization may occur spontaneously. The nuclei of a heterokaryon may divide mitotically of a number of generation.

5. Fig 1. Heterokaryon formation by various mechanisms and heterokaryon segregation. Three distinct types of incompatibility systems are widely distributed among fungi. The incompatibility systems differ in the number of genetic loci involved and also in the number of alleles occur in each locus. Gene controlled the process of mating is called mating type factor, gene or locus. 1) Uni-factor (bi-polar) heterothallism. Uni-factor heterotahllism is controlled by one (A and a or + (plus) and– (minus)) to many alleles (A1, A2, A3, An) in one locus. The mating types segregate into two spore types (Fig 1). Bipolar heterothallism occurs in members of Mucorales, several ascomycetes and many rust fungi. 2) multi-factor (tetra-polar). Multi-factor heterothallism is controlled by many factors in two loci (A and B). The mating types segregate into four spore types (Fig 2). Tetrapolar heterothallism occurs in the majority of basidiomyctes and also the smut fungus. It has been estimated that about 65% of the species having tetra-polar incompatibility mechanism. In certain fungi, for example Schizophyllum commune, if crosses are made between hyphae bearing either common A or common B factor, heterokaryons are rarely formed If the heterokaryon forms has common A, but dissimilar Bs, the reaction is called flat (Fig 2). The growth of this heterokaryon is sparse, depressed, and with knotted, irregularly branched hyphae. The common Bs reaction is called barrage (B) (Fig 2). The hyphae of mating colonies grow towards each other, but at a closer contact zone, the growth ceased leaving a trip separating the colonies. Common Ab heterokaryon has been found to occur. In this case, colonies grow over each other.

6. A1B1A1B2 FL A2B1A2B2 -B+ A1B1 FL-+B A1B2 B+-FL A2B1 +BFL- A2B2 A1A2A3A4 -+++ A1 A2+-++ ++-+ A3 +++- A4 Aa -+ A +- a Fig.1 mating reactions of bi-polar fungus Fig 2. Mating reactions of tetrapolar fungus Fig 3. Compatible reaction (a) intermingling between subcultures from the same mycelium (above) and sibs of non-outcrossing Stereum hirsutum (below) and (d) two mating compatible sibs of Stereum sp. (left and right), incompatible reaction (b) rejection between non-sibs of non-outcrossing S. sanguinolectum c) inhibition in a bow-tie shape sibs of S. hirsutum, e) localized pattern of secondary mycelium establishment in a region of rejection.

7. Fig 4. Rejection with pigmented droplet exudation (a), line gap (b) and inhibition (c), among intergeneric mating The response of incompatibility may vary from line gap (Fig 3b and c, Fig 4 b), inhibition (Fig 3 e and Fig 4 c) and exudation (Fig 4a) Parasexual cycle Parasexual cycle become a source of variation of asexual fungi. Parasexual cycle involve heterokaryosis, diploidization and haploidization. Haploidization involves several mitotic divisions of diploid nuclei. Frequently, daughter nuclei resulting from mitosis has unequal number of chromosomes because sister chromatids failed to separate (non-disjunction) during anaphase. This yields a daughter nucleus with one extra chromosome (2N+1), while the other one chromosome less (2N-1). These nuclei are aneuploids (Fig 5), as they do not have even chromosome. The deficient chromosome nucleus will undergo additional loss chromosome in subsequent mitosis until reduced to haploid condition. Parasexual cycle may be accompanied by mitotic crossing over (Fig 6). In mitotic crossing over, segment between homologous chromosomes are exchange for exactly corresponding segments in homologous chromosomes. Mitotic crossing over are often occurs during haploidization. The probability of recombination from mitotic crossing over is about 10%. The product of parasexual cycle is nonsexual spores, which differ genetically from the parent mycelia. Although the parasexual is mimicry of sexual cycle, but the parasexual cycle is less efficient in recombination production comparing to sexual cycle. Mitotic crossing The recombinationin parasexual cycle is lower than meiotic crossing over. number will increase in subsequent reproduction.

8. Fig 5. Haploidization process causing formation of aneuploidy. Fig.6. Mitotic crossing over between a single pair of homologous chromosome during haploidization, a) diploid nucleus that is heterozygous for genes paba (p- amino benzoic acid-requiring)nand bi (biotin requiring), b) crossing over and c) genetically different nuclei formed after chromosome segregation. Cytoplasmic Inheritance Phenotype of dikaryon and the successfulness of mating are not only controlled by nuclear gene, but also by gene occurring in the cytoplasm. The inheritance controlled by extranuclear gene is called cytoplasmic inheritance. The inheritance of the character determined by these genes donot follow the Mendelian pattern.Traits controlled by cytoplasmic inheritance will not segregate with nuclei but instead inherited from one parent.

9. In fungi, cytoplasmic inheritance dealt with transferring viral or plasmid nucleic acids or mitochondrial DNA. For example Poky in Neurospora crassa and petite Saccharomyces cerevisiae and senescence of Pseudospora anserine.Poky in Neurospora crassa is characterized by slow growth colony. Poky is resulted from deletion of four base pairs in mDNA, causing the production of ribosomal RNA lacks 38 to 45 nucleotides and result in the defiency of a small ribosomal unit. These impairs the synthesis of protein in the mitochondria. In a pocky strain cytochromes A dan B are high, while C is low.Senescence in Podospora anserina is characterized by decline vigour, or further cease growing. These caused by respiration impairements which is actually determined by the occurrence of mitochondrial. Hypovirulency in Cryphonectria parasitica and Saccharomyces cerevisiae strain Killer are determined by the presence of dsRNA. Questions: 1. What is the difference between parasexual and sexual reproduction in term of recombination production effectivity? Please explain in less than 6 sentences. 2. What is aneuploid? Referrences: Ainsworth, AM. 1987. Occurance and Interaction of outcrossing and non-outcrossing populations in Stereum, Phanerochaete and Coniophora in Rayner,ADM, CM Brasier and D. Moore (eds).Evolutionary Biology of the Fungi. Cambridge: Cambridge University Press. p. 285-300. More-Landecker, E. 1996. Fundamentals of the Fungi. 4 th ed. New Jersey: Prentice Hall.