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A/a incompatibility in Neurospora crassa : novel suppressors and nuclear incompatibility Vellani, Trina Sehar 1991

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A/a INCOMPATIBILITY IN NEUROSPORA CRASSA—NOVEL SUPPRESSORS AND NUCLEAR INCOMPATIBILITY by TRINA SEHAR VELLANI B.Sc.(Hon.)# McMaster University, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 (c) Trina Sehar Vellani, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany The University of British Columbia Vancouver, Canada Date -3 OrfnW 1QQ1 DE-6 (2/88) ABSTRACT The sexual functions of the mating type gene (int) of Neurospora crassa include specification of mating identity (Shear and Dodge, 1927) and perithecial maturation (Griffiths and DeLange, 1978; Staben and Yanofsky, 1990). The gene also acts as a vegetative incompatibility locus, so that A + a heterokaryons (Beadle and Coonradt, 1944) or A/a duplication strains (Newmeyer and Taylor, 1967) grow poorly or not at all. An intriguing question regarding the mating type gene is this: How does it control both the switch between somatic and meiotic events and heterokaryon incompatibility? Several research groups (Glass, et al., 1990; Staben and Yanofsky, 1990) are presently studying the sexual functions of the mating type genes. I present a study of the incompatibility function. Two experiments were performed. The first was a search for new suppressors of mating type-associated incompatibility, which resulted in the identification of seven new suppressors, none of which was allelic with the one known suppressor, tol. The second was the comparison of growth rates of a mating type mutant (fertile, heterokaryon compatible) in a mixed mating type heterokaryon and in a mixed mating type duplication to determine whether or not cytoplasmic incompatibility is separable from nuclear iii incompatibility. The results obtained suggest that the mating type mutant, am33, eliminates heterokaryon incompatibility without eliminating nuclear incompatibility. The search for suppressors was attempted in order to define more of the genes involved in A/a incompatibility. The analysis of heterokaryon versus nuclear incompatibility was done to investigate the cellular interactions involved in A/a incompatibility. iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES V LIST OF FIGURES vi ACKNOWLEDGEMENT viiGENERAL INTRODUCTION ; 1 Life Cycle 1 Mating Type Gene Functions 4 Mating and Incompatibility in Other Fungi. 6 Mating and Incompatibility in Other Kingdoms 16 INTRODUCTION 1 8 A Suppressor of A/a Incompatibility, tol .18 MATERIALS AND METHODS 22 Strains and MarkersAscospore Isolation 24 Construction of Tester Strain (T(I->II) 39311, ser, trp, tol, a) 25 RESULTS 1 * 28 DISCUSSION 1 51 What is tol? 6 INTRODUCTION 2 62 RESULTS 2 7 DISCUSSION 2 9Molecular Model 101 REFERENCES 103 V LIST OF TABLES Table 1 Strains from Experiment Set 1 23 Table 2 Phenotypes of F2 Strains 38-39 Table 3 Phenotypes of F3 Strains 44 Table 4 Mating Types of F3 Strains 6 Table 5 Mating Types of Hyphal Tips of A/a Compatible F3 Strains 49 Table 6 Summary of Results 1 50 Table 7 Strains and Media Used in the Measurement of Growth Rate of Am64 in a Mixed Mating Type Heterokaryon 68 Table 8 Strains and Media Used in the Measurement of Growth Rate of am33 in a Mixed Mating Type Heterkaryon 71 Table 9 Genotypes of am33 Strains 73 Table 10 Mating Types of Progeny 7 Table 11 Phenotypes of Progeny 79 Table 12 Slopes of Growth Rates of Progeny 95 Table 13 Mating Types of Single Conidial Isolates of Progeny 96 vi LIST OF FIGURES Figure 1 Life Cycle of 2V. crassa 2 Figure 2 Example of Mitotic Crossover 7 Figure 3 Construction of Tester Strain 26 Figure 4 Summary of Selection Protocol for Suppressors..29 Figure 5 First Cross 31 Figure 6 Phenyotypic Classes of Escaped Fl Strains......34 Figure 7 Second Cross 36 Figure 8 Examples of Mitotic Double Crossovers 41 Figure 9 Third Cross 42 Figure 10 Alternative Pairing Hypothesis 53 Figure 11 Mating Type Regions of N. crassa 63 Figure 12 Segregation of Am64 ORF 66 Figure 13 Growth Rate of Am64 in a Mixed Mating Type Heterokaryon 69 Figure 14 Growth Rate of am33 in a Mixed Mating Type Heterokaryon 72 Figure 15A Cross of am33, ad x T(I->II) 39311, ser-3, A...74 Figure 15B Crosses of Rl-14 or Rl-29 x T(I->II) 39311, ser-3, A 75 Figure 16 Growth Rates of Controls 81 Figure 17 Growth Rates of a-h-x 2 Figure 18 Growth Rates of a-h-x 83 Figure 19 Growth Rates of a-h-x 4 Figure 20 Growth Rates of a-i-x 85 Figure 21 Growth Rates of a-i-x 6 vii Figure 22 Growth Rates of 14-h-x 87 Figure 23 Growth Rates of 14-h-x 8 Figure 24 Growth Rates of 14-i-x 89 Figure 25 Growth Rates of 14-i-x 90 Figure 26 Growth Rates of 29-h-x 1 Figure 27 Growth Rates of 29-h-x 92 Figure 28 Growth Rates of 29-i-x 3 Figure 29 Growth Rates of 29-i-x 94 viii ACKNOWLEDGEMENT I am immensely grateful to the following people: Tony Griffiths for his support, for fostering my scientific independence and for allowing me freedom to develop my own ideas; Louise Glass for her encouragement and countless hours of critical discussion; Jim Berger for his boundless enthusiasm for the world of science and for keeping me from straying too far from the task at hand; Carolyn Myers for sharing her research ideas and for invaluable technical advice; Rod, Mishu and my parents for believing in me. 1 GENERAL INTRODUCTION This work is a two-part investigation of the vegetative incompatibility function of the mating type gene of Neurospora crassa. The first part describes the generation of suppressors of A/a incompatibility and the second part describes the analysis of incompatibility on a cellular level. The mating type gene is involved in both the vegetative and sexual phases of the life cycle. Life Cycle W. crassa, a mold that grows at the sites of recent fires and in decaying vegetation, is a heterothallic ascomycete. Its life cycle is shown in Fig. 1. The haplold, partially septate mycelia grow from ascospores which are germinated by a period of heating. Growth requires the presence of inorganic salts, biotin and a utilizable carbon source (Fincham, et al., 1979). Several days after germination, the hyphal tips begin to delimit two types of conidia which, upon germination, are able to initiate new mycelia. Macroconidia are oval and multinucleate; whereas microconidia are spherical and binucleate or uninucleate (Fincham, et al, 1979). Low levels of nitrogen initiate the sexual cycle. Male structures—conidia and vegetative hyphae—are already present in each single mating type colony (A or a), and 2 oscogenous hypho igure 1 Life cycle of N. crassa (from Fincham, et al., 1979). 3 immature female structures—protoperithecia—begin to form from the same mycelium. The outer layer of the protoperithecium is a wall of hyphae. Inside is a coiled hypha, the ascogonium, from which project female reproductive hyphae, trichogynes. Although each mycelium is hermaphroditic, it is also self-sterile, and sexual fusion between male cells and protoperithecia can occur only between individuals of different mating types. Once fertilized, the protoperithecium is known as a perithecium (Fincham, et al., 1979). Conidia emit a pheromone which directs trichogynes to grow toward them (Bistis, 1981; 1983). When contact is made between the male and female cells, plasmogamy ensues. The male nuclei, presumably under their own genetic control (Vigfusson, et al., 1971), travel down the trichogyne, where one nucleus (Sansome, 1949) enters the ascogonium and becomes associated with the female nucleus. While the perithecium darkens and enlarges, a series of synchronous nuclear divisions gives rise to a cluster of dikaryotic ascogenous hyphae (Fincham, et al., 1979). Karyogamy occurs in the penultimate cells of the ascogenous hyphae, followed directly by meiosis plus two rounds of mitosis. Several days later, the ascospores have become multinucleate (Raju, 1980). The final products of the sexual cycle are perithecia containing many mature asci, each housing eight ascospores which are shot through an opening in the perithecial beak (Fincham, et al., 1979). 4 Mating Type Gene Functions The mating type genes, A and a, are unusual, even among Neurospora species, in that they control two functions, mating and vegetative incompatibility. A/a incompatibility is not seen in either N. tetrasperma (Dodge, 1935) or N. sitophila (Mishra, 1971). Early attempts to resolve the two functions by recombination failed (Pittenger, 1957; Newmeyer, et al., 1973); although later, Griffiths and DeLange (1978) reported the finding of a mating type mutant (am33) that was heterokaryon compatible, yet fertile. In N. crassa, only strains of opposite mating types are able to cross (Shear and Dodge, 1927); so A x a is a successful pairing, but A x A or a x a is not. Strains of opposite mating types are vegetatively incompatible (Beadle and Coonradt, 1944); so A + A or a + a anastomose to form thriving heterokaryons, and A + a fuse, but the anastomosed area dies (Garnjobst and Wilson, 1956). The protoplasm of the fused, and sometimes surrounding, cells becomes granular or vacuolated. Mixed mating type heterokaryons with varying degrees of vigour can be made using forcing auxotrophic markers (Beadle and Coonradt, 1944; Gross, 1952; DeLange and Griffiths, 1975). Protoplasmic killing, more severe than that seen between A and a, is observed in the reactions between incompatible alleles of the heterokaryon incompatibility genes het-C/c, het-D/d and het-E/e (Perkins, 1974). The 5 killing reaction can be detected in vivo, and also when protoplasm from one strain is microinjected into cells of an incompatible strain, at least between strains of different het-C/c or het-D/d genotypes (Wilson, et al., 1961; Williams and Wilson, 1966). Mixed mating type heterokaryons generally escape from their poor growth and start to grow at wild-type or near wild-type rates by deletion of one or the other of the mating type genes (DeLange and Griffiths, 1975). Heterokaryons heterozygous for the vegetative incompatibility genes het-J/j or het-K/k escape by deletion or mutation of the genes (Pittenger, 1964). Strains with a heterozygous duplication of the vegetative incompatibility gene, het-6, also escape by deletion of one of the genes (Glass, personal communication). Not only are the mating type genes incompatible in a heterokaryon, they are also incompatible in a duplication. Strains carrying a heterozygous duplication of the mating type genes grow poorly due to the presence of opposite mating type genes in one nucleus. A/a duplication strains, called "dark agar" strains, produce a brown pigment when grown on glycerol complete medium and their morphology has been described as being spidery (Newmeyer and Taylor, 1967; Turner, et al., 1969). Other duplications in N, crassa grow normally (provided the duplication does not cover any of the heterokaryon incompatibility genes) and are frequently barren, i.e. they produce abundant perithecia, but few 6 spores (Newmeyer and Taylor, 1967). A/a duplications escape from their inhibited growth and start growing at wild-type or near wild-type rates by the somatic segregation of A from a through mitotic crossing over (Fig. 2) or deletion of one of the mating type genes (Newmeyer and Taylor, 1967). Mitotic crossing over yields a culture, barren due to the presence of duplicated genetic material, that is a mixture of mostly A or a homozygous cells. Such cultures are unstable and tend to be overgrown by one nuclear type. Deletion yields a culture, fertile due to the loss of part or all of the duplication, that is a mixture of A or a hemizygous cells. These cultures are also overgrown by one nuclear type (Newmeyer and Taylor, 1967). Mating and Incompatibility in Other Fungi The majority of research on yeast mating type has been done on Saccharomyces cerevisiae, also known as budding yeast because haploid cells reproduce vegetatively by budding. Mating begins with Gl arrest. Pheromone from a cells, a-factor, arrests alpha cells, and pheromone from alpha cells, alpha-factor, arrests a cells. Pairs of opposite mating type cells fuse, undergo karyogamy and then follow either of 2 paths, depending on nutritional conditions. The a/alpha diploid cells reproduce mitotically ' four+ £ o -• — •-r? ^ o o -r-CO /t o c o T 0. —»—, C * + + 3 £ co O C O 0 . • — • — » — »— #-nicn Example from Newmeyer and Taylor (1967) of mitotic crossover that leads to production of cells homozygous for the mating FiflllTP 9 type genes. The duplication was a product from a cross to an l lyUI C c. inversion strain. 8 unless they are nutritionally deprived, in which case they undergo meiosis (see review by Herskowitz, 1988). The mating types of the haploid cells are specified at the mating type locus, MAT (Lindegren and Lindegren, 1943), which codes for transcription factors that control the expression of genes involved in pheromone production, mating and sporulation. Cells with mating type a have the MATa allele which encodes two polypeptides, al and a2; cells with mating type alpha have the MATalpha allele which also encodes two polypeptides, alphal and alpha2. A portion of the MAT genes, called Ya and Yalpha (Nasmyth, et al., 1981), is specific to a and alpha cells, respectively (Sprague, et al., 1981). Three of the mating type polypeptides, al, alphal and alpha2, are involved in the regulation of a-specific, alpha-specific and haploid cell-specific genes. The function of a2 is unknown (Astell, et al., 1981). In a cells, al is produced (Kassir and Simchen, 1976) and a-specific and haploid specific genes are expressed. In alpha cells, alphal induces the expression of alpha-specific genes (Sprague, et al., 1983) and alpha2 represses a-specific genes (Hartig, et al., 1986; Wilson and Herskowitz, 1984). In a/alpha diploid cells, alpha2 carries out the same function as it does in haploid alpha cells, repressing a-specific genes (Strathern, et al., 1981), but it has an additional role. A combination product of alpha2 and al 9 represses the expression of alphal, and of haploid specific genes and stimulates sporulation (Strathern, et al., 1981). Sporulation begins when a/alpha diploid cells are starved of nitrogen and carbon (Esposito and Klapholz, 1981). The regulatory protein al/alpha2 activates meiosis by blocking the expression of RMEI, an inhibitor of meiosis (Mitchell and Herskowitz, 1986). Strains of S. cerevisiae with the dominant allele of the homothallism gene, HO, are homothallic, whereas strains with the recessive allele, ho, are heterothallic. The HO gene product catalyses high frequency interconversion of mating types (Hicks and Herskowitz, 1976). Mating type interconversion occurs by the switching of the genetic information at the MAT locus (Nasmyth and Tatchell, 1980; Strathern, et al., 1980; Hicks, et al., 1977). The information comes from two transcriptionally silent genes, HMR and HML, that flank the mating type gene. Each locus contains a copy of a or alpha. The two loci are kept silent by the action of unlinked genes called SIR (for "silent information regulator") (Abraham, et al., 1983). The switching process begins with a double-stranded cut at MAT (Strathern, et al., 1982) by the endonuclease encoded by HO (Kostriken and Heffron, 1984). A conversion-like event follows, in which heteroduplex DNA forms between the donor locus (HMR or HML) and MAT. The heteroduplex DNA is repaired using the donor DNA as a template (Klar and Strathern, 1984; Klar et al., 1984). Switching is prevented 10 in diploid cells by the repression of HO (Jensen, et al., 1983). Mating in the fission yeast, Schizosaccharomyces pombe, is similar in some ways to that in Saccharomyces cerevisiae. Haploid cells have the mating type h+ or h~ and propagate vegetatively by fission, not by budding. During mating, which occurs under nitrogen starvation conditions, one cell of each mating type participates in the formation of a diploid zygote. The zygote immediately undergoes meiosis and sporulation (Leupold, 1950 cited in Kelly, et al., 1988). As in homothallic strains of S. cerevisiae, S. pombe regularly switches alleles at the mating type locus (Egel, 1977; Miyata and Miyata, 1981). The mating type complex contains 3 regions—matl, mat2-P and mat3-M—which control conjugation, meiosis and sporulation (Kelly, et al., 1988). Two silent loci, mat2-P and mat3-M donate information to matl which confers mating type, either h+ (matl-P) or h~ (matl-M) (Egel, 1977; Egel and Gutz, 1981; Beach, 1983). The complex has been sequenced (Kelly, et al., 1988). Two genes are encoded by each of matl-P and matl-M, two of which are required for conjugation and specification of mating type, and all 4 of which are required for meiosis and sporulation (Kelly, et al., 1988). Each of matl, mat2-P and mat3-M contains 2 blocks of sequence homology. The silent genes, mat2-P and mat3-M, alone contain a third region of 11 homology which probably acts as a silencer (Kelly, et al., 1988). Like RMEI of S. cerevisiae, the protein, rani, of S. pombe is an inhibitor of meiosis. Its action is blocked by the protein, mei3, which is produced in h+/h~ cells (McLeod and Beach, 1988). Neurospora crassa could be like S. cerevisiae in that the mating type genes could act like al/alpha2, combining to form a transcription factor that blocks the synthesis of a meiosis inhibitor, or it could be like S. pombe in that a product analogous to mei3 could be produced in A/a mating diploid cells. The complex system of regulating cell type in S. cerevisiae must differ in N. crassa because each mating type idiomorph encodes but one transcript. That the combination product, A/a, could act as a transcription factor is supported by the sequence similarity between the a idiomorph and the HMG box motif, a DNA-binding sequence (Staben and Yanofsky, 1990). The A idiomorph has no obvious DNA-binding motif, but it does have similarity to the MATalphal protein of S. cerevisiae and could interact with the a product during mating. During the vegetative state, A identity could be specified by A-specific genes, turned on by the A product interacting with a transcription factor that binds to their promoter regions (Glass, et al., 1990). Strains containing a heterozygous duplication of the mating type region do not lose either their A or a identity, 12 so the combination product does not exclude mating type specificity. Homothallism in W. crassa must also occur by a different mechanism than switching because each strain in the homothallic species N. dodgei, IV. galapagonensis, N. africana and W. lineolata has only one copy of a sequence homologous to the N. crassa mating type gene, A. The homothallic species, N. terricola, has sequences homologous to the N. crassa mating type genes, A and a, but only one copy of each (Glass, et al., 1988). The incompatibility systems, also called breeding systems, of the 2 basidiomycetes, Coprinus cinereus and Schizophyllum commune, are tetrapolar. The mating type complex is comprised of 2 regions, A and B, each containing 2 genes, alpha and beta, with multiple alleles. Mating requires that the 2 participants differ at a minimum of one A gene and one B gene. Close linkage of alpha and beta restricts inbreeding potential by inhibiting recombination (see Koltin, et al., 1972). Cloning of the A factor of C. cinereus has revealed that the alpha and beta regions are themselves composed of a number of genes with multiple alleles, some of which are common to other alleles of A and some of which are unique (E. Mutasa, A. Tymon, W. Richardson, U. Kues and L. Casselton, 1991 in published abstracts from Sixteenth Fungal Genetics Conference). 13 Three alleles of the A-alpha region of S. commune have been cloned, sequenced and shown to contain multiple transcripts, some shared and some unique. Some of the postulated polypeptides contain homeodomain motifs (R.C. Ullrich, M.M. Stankis, H. Yang and CP. Novotny, 1991; G. May, 1991 in published abstracts from Sixteenth Fungal Genetics Conference), implying that the products regulate the expression of other genes. In the pathogenic basidiomycete, Ustilago maydis, dikaryosis between individuals from different incompatibility groups is a prerequisite for pathogenic infection. Different a alleles are required for fusion and different b alleles for pathogenicity. The two alleles of a have been cloned and they encode a product required for mycelial growth, a condition necessary for infection (M. Bolker and R. Kahmann, 1991 in published abstracts from Sixteenth Fungal Genetics Conference). Ten b alleles have been cloned, and subsequent molecular analysis has revealed that they share a homeodomain-related motif, implying that the b polypeptides bind DNA, possibly to regulate sexual development (R. Kahmann, B. Gillissen, R. Schleshinger, C Sandmann, F. Schauwecker, J. Bergemann, B. Schroeer, M. Bolker and M. Dahl, 1991 in published abstracts from Sixteenth Fungal Genetics Conference). Like the A and B regions of S. commune and C. cinereus, the b region of U. maydis is composed of 2 genes, b-east and Jb-west. Null mutants of the b region are mating deficient, suggesting that the postulated b heterodimer formed during mating is an activator of mating genes (Kahmann, personal communication) Studies done with expression of two of the b alleles have identified a 70 amino acid region responsible for allele specificity. These 2 b alleles are constitutively expressed in diploids and haploids, although at a lower level in the latter (L. Giasson, A. Yee and J.W. Kronstad, 1991 in published abstracts from Sixteenth Fungal Genetics Conference). Similarly, in W. crassa the mating type genes are expressed during sexual and vegetative phases of the life cycle, but at a lower level in the latter (Staben and Yanofsky, 1990; Glass, et al., 1990). N. crassa differs from the basidiomycetes in several ways. The mating type genes of W. crassa have only one ORF so unlike the basidiomycetes, they do not function as restrictors of inbreeding. Also, in N. crassa, vegetative compatibility is not a prerequisite for mating. Fusion of hyphae with compatible genotypes leads to heterokaryon formation and fusion of sexual cells with compatible genotypes leads to mating. The heterokaryosis that occurs between vegetative cells is somehow different from the fusion that occurs between the trichogyne and the male cell Mixed mating type heterokaryons on crossing medium do not exhibit the incompatibility phenotype, so perhaps it is the nutritional conditions that dictate whether the mating type genes will initiate incompatibility or mating. The mating type gene of the yeasts is a master regulator of other genes. The mating type genes of the basidiomycetes and of N. crassa are also postulated to be regulatory, although the mechanisms of their actions must differ. Each of the four major groups of fungi—phycomycetes, basidiomycetes, ascomycetes and fungi imperfecti—exhibits genetically determined incompatibility (Burnett, 1976). In some cases, e.g. Neurospora crassa (Fincham, et al., 1979), Podospora anserina, under the control of the s locus (Esser 1971), Rhizoctonia solani (Burnett, 1976) and Endothia parasitica (chestnut blight fungus) (Anagnostakis, 1977), the incompatibility only affects vegetative heterokaryosis; whereas in others, e.g. Aspergillus nidulans (Jinks, et al. 1961; Grindle, 1963a; 1963b), Podospora anserina, under the control of the a,Jb,c,v loci or non-allelic system (Esser, 1971; Blaich and Esser, 1971), Coprinus lagopus, Schizophyllum commune (Fincham et al., 1979), Ustilago sp. (Fincham et al., 1979; Day and Cummins, 1981) and yeasts (Crandall, 1978), the genetic restrictions on fusion affect fertility. Two species of fungus other than N. crassa that have mating type genes that act as heterokaryon incompatibility loci are Ascobolus stercorarius (Bistis, personal communication) and Aspergillus heterothallicus (Kwon and Raper, 1967). 16 Mating and Incompatibility in Other Kingdoms Each of the five kingdoms of organisms—Monera, Protista, Fungi, plants and animals—shows examples of genetically controlled, intraspecific, sexual or somatic incompatibility. Moreover, interactions between species, for example, those between host and pathogen or between symbionts, can exhibit incompatibility. Sexual incompatibility limits mating between certain individuals within a species. Somatic incompatibility limits fusion between or co-existence of certain cells. Some of the fungi demonstrate incompatible reactions which are both somatic and sexual, in that a successful interaction between seemingly vegetative cells leads to mating. In the kingdom Monera, mating in Escherichia coli requires recognition of mating types; and only pairings between cells of different mating types are compatible (Hayes, 1952; Lederberg, 1957). Ciliates, in the kingdom Protista, generally mate with cells of a different mating type and fusion occurs between "vegetative" cells (there are no cells specialized for mating) under certain environmental and physiological conditions (Nanney, 1977; Ricci, 1981). In plants, there are examples of both sexual and somatic incompatibility. Some angiosperms have the S locus which controls sexual compatibility, allowing pollen to 17 fertilize only females with different S alleles (Lewis, 1954; Ebert et al., 1989; Haring et al., 1990). Somatic incompatibility occurs between different species of plants when tissue from one species is grafted onto an individual of another species, even from the same family (Yeoman et al., 1978). Fusion of cells in the sexual cycle of Chlamydomonas sp. requires recognition of opposite mating types (Wiese and Wiese, 1978; Harris, 1989) which are determined by two alleles, mt+ and Bit"*, at one locus. The alleles are believed to encode proteins that control the expression of other genes or the activity of their products, possibly by forming or causing the formation of a novel regulatory product upon cell fusion (Ferris and Goodenough, 1987). A somatic interaction is seen in vertebrates when a graft of tissue or an organ transplant is rejected from the recipient. The reaction, in this case, is controlled by genes of the major histocompatibility complex (MHC), which are expressed in the T cells of the immune system (see reviews by Klein, 1976; Bach and vanRood, 1976). Humoral recognition occurs in the blood, and it is governed by antibodies specific for the ABO blood group antigens and the Rh factor protein (see review by Katz, 1978). 18 INTRODUCTION 1 A Suppressor of A/a Incompatibility, tol Newmeyer (1970) found a recessive suppressor of A/a incompatibility, unlinked to mt, which she called tol for "tolerant". The new gene had no demonstrable effect on the ability of a strain to mate. It appears to be inactive during starvation because A + a heterokaryons on crossing medium do not exhibit the incompatibility phenotype. However, Johnson (1979) suggested that the gene does have a role during mating because the recessive allele, tol, suppresses fmf-1, a gene specifying female and male fertility. Crosses between fmf-1, tol+ and fmf-l+, tol+ are sterile, whereas crosses between fmf-1, tol and fmf-l+, tol are fertile. Johnson hypothesized that tol permits promiscuous fusion between A and a during mating, allowing transfer of fmf-l+ product from the fmf-l+ strain, resulting in rescue of the sterility phenotype. If tol+ is expressed during the sexual cycle, fusion of sexual structures occurs despite the presence of tol+. It is possible that the cytoplasm of trichogynes is modified to allow the presence of male nuclei of the opposite mating types. Genes that suppress A/a incompatibility will be useful in analysing the mating type gene itself and in deciphering the process of incompatibility. Moreover, if a suppressor has its own phenotype, in addition to suppressing A/a 19 incompatibility, then other functions related to incompatibility may be revealed. For example, if a suppressor prevents incompatibility by altering cell wall structure so that it can no longer be broken down by the incompatibility reaction, it may produce a second phenotype of abnormal morphology. Two types of suppressors, extragenic and intragenic, are discussed below. Extragenic suppressors probably interact with the mating type genes; and their detection will help in the dissection of the mechanism of incompatibility. Theories as to how tol affects incompatibility are considered in Discussion 1. Other suppressors, besides tol, have been identified— two found in nature and two induced in the laboratory. Smith and Perkins (1972) noted that the osmotic-sensitive, reciprocal translocation strain, cut, suppressed A/a incompatibility. Newmeyer (1970) reported that the wild type strain, Panama a, (Fungal Genetics Stock Center #1132) segregated compatible and incompatible progeny when crossed to a duplication-generating inversion A tester. It is unknown whether or not this suppressor is allelic with tol. Newmeyer (1970) found another suppressor, which may or may not be allelic with tol, in an escaped A/a duplication strain (called N83) from a cross between an inversion A strain and a normal sequence a strain. DeLange and Griffiths (1975) reported that 2 of their escaped mixed mating type heterokaryons, in which one component was tol 20 and the other tol+, produced perithecia with both mating type testers, but produced ascospores with only 1. They suggested that these 2 strains arose by deletion or lethal mutation at tol+. Intragenic suppressors and mutants of the mating type genes will help, when mapped and sequenced, to reveal the part(s) of the mating type genes governing vegetative incompatibility. Especially useful will be those mutants in which only one of the mating type functions is defective, e.g. the fertile, heterokaryon compatible mutant, am33. When Newmeyer discovered tol, she was using strains of N. crassa with A/a duplications, dark agars. They were meiotic segregants from a cross of a normal sequence A to a strain of mating type a carrying a pericentric inversion of a large portion of linkage group I (L.G. I) (Fig. 2). Most of the dark agars escaped from their inhibited growth by the somatic segregation of A from a. One strain, however, escaped by mutation at tol, thereby allowing both mating type genes to reside within one nucleus without causing vegetative incompatibility. The new tol gene proved to be a suppressor, not only of nuclear incompatibility, but also of heterokaryon incompatibility, allowing the vigorous growth of both duplicated A/a,tol strains and A,tol + a,tol heterokaryons. Newmeyer favoured the use of a A/a duplication to cause inhibited growth because it eliminated the possibility that incompatibility was due to alleles at some other 21 heterokaryon incompatibility locus. Furthermore, if a mixed mating type heterokaryon had been used, only "dominant" alleles of suppressor genes would have allowed escape to occur. A method similar to that used by Newmeyer (1970) has been used to generate novel suppressors of A/a incompatibility. The search is an attempt to chart the genetic interactions involved in mating type-associated i ncompat i bi1ity. 22 MATERIALS AND METHODS General protocols were standard, and are described in Davis and DeSerres (1970). Crosses were made at 25°C in 15 cm test tubes or, if fl females were used, on petri plates. Ascospores with A/a duplications were selected as meiotic segregants from crosses to strains in which the mating type gene had been translocated from L.G. I onto L.G. II. Duplication progeny contained L.G. II from the translocation parent and L.G. I from the other parent. This combination of linkage groups was selected as follows. The mating type genes were marked, one with an auxotrophic marker (ser-3) and the other with a temperature sensitive marker (un-3), and ascospores were plated on minimal medium at 32°C which permitted survival of only the duplication spores. Strains and Markers A list of strains and their sources is shown in Table 1. Strains were maintained at room temperature on standard media. The marker, un-3, is temperature-sensitive, with strains growing poorly between 28.5°C and 30°C and not growing at all at temperatures above 30°C. It is located 0.04 to 0.1 map units to the left of the mating type gene (Perkins, et al., 1982). The marker contains two mutations, Table 1 Strains from Experiment Set 1 STRAIN GENOTYPE SOURCE R601 un-3, A C.J. Myers R602 un-3, a C.J. Myers T(l->ll)39311,ser,A T(l->ll)39311, ser-3, A C.J. Myers T(l->ll)39311,ser,a T(l->ll)39311, ser-3, a C.J. Myers T(l->ll)39311,ser,trp,tol,A T(l->ll)39311, ser-3, trp-4, tol, A C.J. Myers T(l->H)39311,ser,trp,tol,a T(l->ll)39311, ser-3, trp-4, tol, a This work trp,tol,A trp-4, tol, A N.L. Glass (F.G.S.C.* #2336) fl,A F.G.S.C. #4960 fl,a F.G.S.C. #4961 •F.G.S.C.-Fungal Genetics Stock Center 24 separated by 0.1 map units, one in cytochrome-20 and the other in ethionine-2 (A.M. Lambowitz, unpublished results, Griffiths, personal communication). Tests for un-3 were done on minimal medium at 25°C and at 37°C and were repeated 3 times each for positive identification. In these tests, a higher temperature (37°C) could be used than was used to select duplication progeny (32°C) because the un-3 phenotype is more obvious at 37°C and it was not necessary to avoid killing the fungi. The translocation T(I->II) 39311 is an insertion of an interstitial segment of the left arm of linkage group I (including ser-3, un-3 and mt) into the right arm of linkage group II, inverted with respect to the centromere (Perkins, 1972). The gene, ser-3, specifies a requirement for serine and is located fewer than 2 map units to the left of the mating type gene (Perkins, et al., 1982). The gene, trp-4, specifies a requirement for tryptophan and is linked to tol by less than 1 map unit (Perkins, et al., 1982). The aconidial mutant, fluffy (fl), is highly fertile and is used as a mating type tester. Ascospore Isolation Ascospores were isolated as follows, unless stated otherwise. Spores were collected from the sides of the 25 crossing test tubes with a wire loopful of sterile distilled water and put into eppendorf tubes of sterile distilled water or 0.1% agar. Hemocytometer counts were done to determine the necessary dilution factor that would yield approximately 10-30 spores per plate. Diluted spores (1/4 mL) were pipetted and spread onto sorbose plates with a bent glass rod. The plates were placed into a 60°C oven for 30 minutes to heat shock the spores to initiate germination. Spores were left at room temperature for 4-5 hours to ensure that the mycelia were well established prior to selection. The plates were left overnight in a 32°C incubator so that the colonies grew large enough to be seen and harvested. Individual germinated spores were collected by cutting out a square of agar containing the mycelium and placing it into a test tube containing 1 mL of Westergaard and Mitchell's liquid medium. If, after several days, the level of the liquid in the tubes dropped below half, then sterile distilled water was added to maintain the level of liquid at 1 mL. Construction of Tester Strain (T(I->II) 39311, ser, trp, tol, a) The strain, T(I->II) 39311, ser, trp, tol, a was made from a cross of a female trp, tol, A to a male T(I->II) 39311, ser, a, from which ser, trp, tol, a progeny were selected (Fig. 3). The female was inoculated onto synthetic 26 T(l->ll)39311,ser,a x trp,tol,A ser a II trp tol IV IV select X II trp tol IV IV dead ser a II trp tol IV IV A/a II trp tol IV IV Construction of the strain T(l->ll)39311,ser,trp,toi,a. The strain, marked X, was a product of the cross between T(l->ll)39311,ser,a x trp,tol,A 27 crossing medium containing 10% of the normal concentration of tryptophan (i.e. 20 mg/L instead of 200 mg/L) because the cross was sterile on the normal concentration of tryptophan. Presumably the tryptophan was providing too much nitrogen for the sexual cycle to be initiated (Myers, personal communication). 28 RESULTS 1 An overall scheme of the procedure used in generating the suppressors is shown in Fig. 4. Duplication strains (Fl) were selected as meiotic segregants from a set of crosses (referred to as the "first cross") between a normal chromosomal sequence parent and a translocation parent. The duplication strains escaped from inhibited growth (escaped Fl) and were tested for their mating types. Those which retained both mating types (A/a escaped Fl) possibly contained the desired suppressors and were crossed to normal chromosomal sequence fl strains to remove the translocation chromosome. This set of crosses is called the "second cross". Some of these F2 progeny presumably contained suppressors. Only the temperature-sensitive F2 strains were tested for the presence of suppressors because the un-3 marker was needed to select duplication progeny in the third set of crosses. The temperature-sensitive F2 strains were crossed to tester strains containing the translocation T(I->II) 39311. Duplication progeny were selected and assessed for their compatible/incompatible phenotypes. Detailed descriptions of each step follow. Duplication strains (labelled "Fl" in Fig. 4) were created (Perkins, 1972) from a pair of crosses, reciprocal in the sense that the mating types were reversed. This set of crosses is referred to as the "first cross" and is 29 First Cross (to generate A/a duplications): R601,un-3 x T(l->ll)39311,ser,a minimal medium, 32 degn es to select duplications F1 A/a Duplication Strains escape to select suppressors Escaped F1 Strains Choose A/a Escaped F1 (presumed suppressor strains) Second Cross (to eliminate duplication): A/a Escaped F1 x fl,a and fl,A F2 Choose temperature sensitive (t.s.) F2 (need un-3 for selection of duplications in third cross) Third Cross (to confirm suppressor phenotype): t.s. F2 x T(l->ll)39311,ser,trp,tol minimal medium, 32 degree t to select duplications F3 (t.s. F2 strains that produced incompatible and compatible duplication F3 progeny were the suppressor strains) —. . A summary of the selection protocol for suppressors. The llCjUre 4 reciprocal cross was R602 x T(l->ll)39311,ser,A. 30 described in the following three paragraphs. One cross in the pair, shown in Fig. 5, was made between a normal chromosomal sequence female parent (R601) and a male parent containing a translocation (T(I->II) 39311, ser, a). In the reciprocal cross (not shown), the normal chromosomal sequence female parent was R602 and the male translocation parent was T(I->II) 39311, ser, A. The normal sequence parents, R601 and R602 had their mating type genes, A and a, respectively, marked with the temperature-sensitive gene, un-3, which is less than 1 map unit to the left of mt. The translocation strains had their mating type genes marked with the auxotrophic gene, ser-3, which is less than 2 map units to the left of mt. The mating type genes were marked to allow selection of progeny that contained both A and a. The germination of spores on minimal medium at 32°C selected progeny that contained L.G. I from the normal parent (ser-3+, un-3, A or ser-3+, un-3, a) and L.G. II from the translocation parent (ser-3, un-3+, a or ser-3, un-3+, A). The genotype of the selected spores was ser-3+, un-3, A/ser-3, un-3+, a or, from the reciprocal cross, ser-3+, un-3, a/ser-3, un-3+, A. Since the nuclei of the selected Fl strains contained both mating types, growth was inhibited and the cultures grew with the dark agar morphology of short hyphae growing in a tight knot (Perkins, 1972). Figure 5 depicts only one possible pairing—that of the two L.G. I's. Although it is not shown in the figure, the 31 R601 T(l->ll)39311,ser,a minimal medium, 32 degrees to select duplications II DIES Ser-3+ Un~3 A ^ f (temperature-sensitive) ser-3 un-3+ a ser-3 un-3+ a II LIVES (A/a duplication) DIES II (deletion) II DIES (serine-requiring) Figure 5 The first set of crosses was done to generate A/a duplication strains. The crosses were R601 x T(l->ll)39311,ser,a and x T(l->ll)39311,ser,A. 32 translocated portion of L.G. I is long enough to pair with L.G. I (Metzenberg, personal communication). As explained in the following paragraph, the occurrence of this pairing pattern would not have affected the experimental design. If the translocated section of L.G. I had paired with the intact L.G. I and a single crossover had occurred in the paired region, the products would not have survived because of the formation of dicentric and acentric chromosomes. If a double crossover had occurred in the paired region, the surviving progeny would only have had one mating type, and therefore would have grown as wild type. These types of spores would not have been chosen as one of the A/a escaped FI strains. Spore cultures with standard incompatibility and compatibility phenotypes were needed as controls for comparison to the duplication progeny. Such strains were obtained as progeny from the cross between the normal chromosomal sequence female, R601 (un-3, A), and the translocation male, T(I->II) 39311, ser, trp, tol, a. Spores were germinated and grown on medium containing tryptophan at 32°C. These conditions selected progeny containing L.G. I (ser-3+, un-3, A) from the female parent and L.G. II (ser-3, un-3+, a) from the male parent. Half of the progeny, those containing L.G. IV (trp-4+, tol+) from the female, were incompatible duplications, and half, those containing L.G. IV (trp-4, tol) from the male were compatible duplications. 33 From the duplication-generating crosses, R601 x T(I->II) 39311, ser, a and R602 x T(I->II) 39311, ser, A, 182 duplication ascospores were collected and maintained on liquid medium. Each duplication culture exhibited the same phenotype as that shown by the A/a, tol+ incompatible controls, growing as a small dense mass of hyphae at the bottom of the test tube. The A/a, tol compatible controls grew faster, filling the test tube after several days. All of the spore cultures escaped from incompatibility within 2 weeks, although escape occurred at different times for each strain. Escape was detected as a shift in hyphal morphology from the dense growth to less dense growth and by an increase in growth rate. In order to eliminate strains that had escaped by deletion of mating type genes and to identify double mating type strains that may have escaped because of mutation to tol or to a tol-like suppressor (A/a escaped Fl strains), the mating type of each escaped Fl strain was tested by spotting it on protoperithecial lawns of fl,A and fl,a testers. The crossing behaviours of the escaped Fl strains allowed their division into 9 phenotypic classes, 8 of which are described in Fig. 6. The one class not shown in the figure was comprised of 25 strains that retained the capacity to cross and produce ascospores with testers of both mating types. These 25 strains (labelled "A/a escaped Fl" in Fig. 4) were the ones presumed to contain REACTION WHEN CROSSED TO fl,A fl,a Legend No reaction Barren perithecia Mature perithecia 6 Eight phenotypic classes of escaped F1 strains that were no used in the experiment. 35 suppressors. The remaining 159 strains were not used in the rest of the experiment, but their possible origins are considered in Discussion 1. The next set of crosses (referred to as the "second cross") was done to demonstrate that the 25 A/a escaped Fl strains actually contained suppressors. Escape in these strains had not occurred by deletion of either of the mating type genes, but it may have occurred by mitotic crossover or by some as yet unidentified mechanism other than mutation or deletion at a new suppressor locus. At the same time, the second cross served to get the suppressors into a stable background, one without duplications. The translocation chromosome, L.G. II, was removed by crossing each A/a escaped Fl culture to fl, a and fl, A females (Fig. 7). Crosses involving a duplication strain are frequently "barren", i.e. they produce few ascospores (Newmeyer and Taylor, 1967). Some of the crosses of the A/a escaped Fl strains to fl females produced as few as 1-6 ascospores. Spores from these crosses were not collected by the plating method because many would have been lost through the procedure. Instead, they were collected individually with a tungsten needle under the microscope and placed in liquid medium. The tubes of liquid medium were heat shocked in a 60°C water bath to initiate germination of the spores, then left at room temperature. The test tubes were checked daily for the appearance of growth and the results are shown Temperature-sensitive II NO ser-3+ un-3 A I ser-3 un-3+ a -Some compatible -Some incompatible YES II ser-3+ un-3+ a ser-3 un-3+ a NO -Some compatible -Some incompatible II NO The second set of crosses was done to eliminate the duplication from the putative suppressor strains. The crosses were: A/a escaped F1 derived from R601 x fl,a and A/a escaped F1 derived from R602 x fI,A. Temperature-sensitive progeny were chosen because un-3 was FlGlJTP 7 needed as a marker in the third cross and to ensure that the iv^uic; l translocation chromosome was eliminated. 37 in Table 2. Despite careful collection of spores, there were 10 crosses from which no spores could be collected. Progeny from these crosses (labelled MF2" in Fig. 4) were either incompatible duplication, compatible duplication or single mating type strains. The compatible and single mating type progeny were subcultured onto minimal medium slants so that sufficient mycelia could be grown for use in subsequent tests. Incompatible progeny were discarded for two reasons—they were duplications and they did not contain suppressors. Compatible and single mating type F2 strains were tested for un-3 because the marker was needed in the next cross. Furthermore, non-duplication progeny were needed for the next cross, and so compatible duplications had to be eliminated. They were discarded with the rest of the non-temperature-sensitive strains. Only 18 F2 progeny out of 329 tested were temperature-sensitive. Possible reasons for the dearth of temperature-sensitive F2 strains are presented in Discussion 1. The 18 temperature-sensitive F2 strains were crossed to the tester strain, T(I->II) 39311, ser, trp, tol, to confirm the presence of suppressors in the former. The temperature-sensitive F2 progeny may not have contained suppressors for a number of reasons. Firstly, in the second cross, L.G.II(T I->II) was removed, so if the mutation had occurred on this chromosome, and had not recombined onto the homolog, it would be lost. 38 Table 2 Phenotypes of F2 Strains STRAIN NUMBER OF F2 STRAINS WITH THE PHENOTYPE NORMAL (INCLUDES COMPATIBLE) INCOMPATIBLE BLANK A1-29 4 2 25 a1-29 3 0 10 A1-54 0 0 4 a1-54 19 8 39 A1-58 1 0 8 a1-58 13 2 25 A1-59 2 0 4 a1-59 20 2 18 A1-65 0 0 1 a1-65 15 4 21 A1-73 12 1 12 a1-73 2 4 19 A1-75 2 0 5 a1-75 5 1 20 A1-103 0 0 0 a1-103 17 14 0 A1-104 0 0 0 a1-104 17 0 23 A1-107 0 0 2 a1-107* 1 1 0 21 A1-113 1 2 9 a1-113* 14 0 36 A1-128 0 0 0 a1-128* 19 8 40 A2-18 0 0 3 a2-18 21 6 14 A2-20 1 0 13 a2-20 9 2 8 A2-32 3 0 10 a2-32 14 4 15 A2-37 3 2 23 a2-37 3 3 19 ..continued Table 2 continued 39 STRAIN NUMBER OF F2 STRAINS WITH THE PHENOTYPE NORMAL (INCLUDES COMPATIBLE) INCOMPATIBLE BLANK A2-57 0 0 7 a2-57 1 1 8 A2-79 0 0 0 a2-79 0 0 0 A2-86* 11 1 0 a2-86 8 0 0 A2-95 0 0 0 a2-95 0 0 0 A2-110 0 0 0 a2-110 0 0 0 A2-117 0 0 0 a2-117 35 2 23 A2-146 2 2 16 a2-146 5 1 5 A2-155* 7 2 41 a2-155 19 4 27 A2-162 0 0 5 a2-162 10 23 17 KEY FOR STRAIN NOMENCLATURE A or a • crossed to fI,A or fl.a in the second cross 1 or 2 • crossed to R601 or R602 in the first cross final # » isolate # from first cross •strains that had temperature-sensitive progeny 40 In this case, all of the duplication progeny from the crosses of temperature-sensitive F2 strains to T(I->II) 39311, ser, trp, tol would be incompatible. Secondly, because of segregation, the temperature-sensitive F2 progeny may have contained the chromosome homologous to the one with the suppressor mutation. I picked as many ascospores as possible from the crosses of A/a escaped FI to fl females, but, as previously mentioned, some of the crosses produced very few. Finally, if the original escape event had been due to mitotic segregation, there would be no suppressor to be found. Newmeyer and Taylor (1967) did report that their A/a escaped strains were heterokaryons of pure A and pure a nuclei, suggesting that somatic segregation had occurred. In the system they used, there were no selective markers close to mt that would prevent the survival of mitotic crossover or mt deletion products. Figure 8 shows examples of mitotic crossovers and their products, some of which survive the selection conditions. If a temperature-sensitive F2 strain contained a suppressor at a locus other than tol or mt, then the duplication progeny (F3) from the cross to T(I->II) 39311, ser, trp, tol would be of two types—compatible or incompatible. If the new mutation had occurred in the tol gene or in the region of the mating type gene controlling vegetative incompatibility, then all of the duplication progeny would be compatible (Fig. 9). POSSIBLE CROSSOVERS MITOTIC PRODUCTS ser+ un A f X ser un+ a ser+ un A f X ser un+ a ser+ un A ser un+ a ser+ un A ser un+ A ser+ un a ser un+ a ser+ un A ser un A ser+ un+ a ser un+ a ser+ un A ser+ un A ser un+ a ser un+ a Figure 8 Mitotic double crossovers that could occur in duplication strains. Single crossovers result in dicentric and acentric products. Thin lines denote L.G. I. Thick lines denote L.G. II. (N.B. The insertion is inverted with respect to the centromere.) 42 + un-3 A I It IV 1/2 "suppressors" it I ser + a trp tol |V IV "\trp tol |V + un-3 A ser + a IV IV Progeny surviving on minimal medium at 32 degrees IV "\trp tol |V 4f ser Figure 9 IV IV The third set of crosses was done to confirm the suppressor phenotype of the putative suppressor strains. The crosses were: R601-derived suppressors x T(l->ll)39311,ser,trp,tol,a and R602-derived suppressors x T(l->ll)39311,ser,trp,tol,A. If the new mutation had occurred at mt or tol, all of the surviving progeny would have been compatible duplications. If the new mutation had occurred at a new "tol' locus, some of the surviving progeny would have been compatible duplications and some would have been incompatible duplications. 43 Ascospores from the crosses of temperature-sensitive F2 strains to T(I->II) 39311, ser, trp, tol were selected as described in Materials and Methods. Table 3 shows the phenotypes of the F3 progeny from each of the 18 crosses. Seven strains segregated compatible and incompatible progeny. These were the suppressor-containing F2 strains. Ten strains segregated only incompatible progeny. These strains may have contained the chromosome homologous to the one with the suppressor. It is unlikely, however, that all 6 of the F2 progeny derived from al-128 would have received the chromosome homologous to the one with the suppressor. There are two additional (and more likely) explanations for the lack of suppression in these six strains. The original A/a duplication may have escaped by mitotic segregation or by mutation/deletion at a suppressor locus on L.G. II. Since there were no temperature-sensitive F2 strains that segregated all compatible progeny, none of the original escape events could have been due to mutation at either the mating type genes or at tol. One of the strains, al-128-27, although crossed on several occasions to T(I->II) 39311, ser, trp, tol, produced no perithecia or ascospores. The sterility of al-128-27 could have been unique to this cross because the strain was able to induce the production of perithecia when crossed to a female fl, a tester. A sample of the compatible and incompatible F3 strains Table 3 Phenotypes of F3 Strains F2 STRAIN NUMBER OF F3 STRAINS WITH THE PHENOTYPE COMPATIBLE INCOMPATIBLE a1-113-7 0 31 a1-113-8 24 22 a1-113-9 0 6 a1-113-10 0 46 A2-155-2 7 18 A2-155-3 14 51 A2-155-5 18 28 A2-86-1 0 30 A2-86-4 0 36 A2-86-12 9 21 a1-107-9 19 18 a1-107-11 28 12 a1-128-22 0 32 a1-128-23 0 32 a1-128-24 0 25 a1-128-25 0 35 a1-128-26 0 32 a1-128-27 0 0 KEY FOR STRAIN NOMENCLATURE A or a = crossed to fI,A or fl,a in the second cross 1 or 2 - crossed to R601 or R602 in the first cross middle # • isolate # from first cross last # - isolate # from second cross 45 was tested for mating type (Table 4). Almost all of the compatible F3 strains (65 of 66) contained both mating types. This is the class of progeny that show the existence of suppressors. It is unlikely that they grew well due to escape because of the high proportion of A/a strains. Furthermore, the hyphae of escaped strains are wispy, whereas these 65 compatible strains grew with a dense morphology. One of the apparently compatible F3 strains (one of the progeny strains from the R601-derived parent, al-113-8) contained only A. Although escape usually does not occur within the first 24 hours after germination of the spore, it may have escaped earlier than normal from A/a incompatibility by loss of the mating type gene from the translocated segment. It is unlikely that the strain resulted from a double crossover on either side of un-3 because of the close linkage of un-3 to ser-3 and mt. It is also unlikely that the strain survived due to reversion of the un-3 mutant because the un-3 marker contains two mutations. Most of the incompatible F3 strains (59 of 71) only contained one mating type, probably because the mating type tests were done after the incompatible strains had escaped. Those incompatible F3 strains which contained both mating types may have escaped by mutation at a suppressor locus or by mitotic crossing over between the L.G. I centromere and mt and between mt and un-3, and both types of derivative 46 Table 4 Mating Types of F3 Strains TEMPERATURE-SENSITIVE F2 COMPATIBLE F3 INCOMPATIBLE F3 a1-113-8 9 A/a 1 A/a 1A 1 A 8 a a1-107-9 10 A/a 1 A/a 9 a a1-107-11 10 A/a 1 A 9 a A2-86-12 9 A/a 2 A/a 6 A 3 a A2-155-2 7 A/a 3 A/a 5 A 2 a A2-155-3 10 A/a 8 A 2 a A2-155-5 10 A/a 5 A/a 4 A 1 a The presence of suppressors was implied by the production of A/a compatible F3 progeny. KEY FOR STRAIN NOMENCLATURE A or a - crossed to fI,A or fl.a in the second cross 1 or 2 • crossed to R601 or R602 in the first cross middle # • isolate # from first cross last # • isolate # from second cross 47 nuclei were still present. Most of the escaped incompatible F3 strains derived from R601 (un-3, A) were a, and most of the escaped incompatible F3 strains derived from R602 (un-3, a) were A. These two types of strains could have occurred by deletion of mt and un-3 from the temperature-sensitive L.G. I homolog. Another mechanism could have been double mitotic crossing over, between the L.G. I centromere and mt and between mt and un-3, followed by overgrowth by the a nuclei in the R601-derived a escaped strains or by the A nuclei in the R602-derived A escaped strains. The latter mechanism could have given rise to the smaller classes of escaped strains, R601-derived A escaped strains and R602-derived a escaped strains. These classes could have arisen by mitotic crossing over between the L.G. I centromere and mt and between mt and un-3, followed by overgrowth by the A nuclei in the R601-derived A escaped strains or by the a nuclei in the R602-derived a escaped strains. Although the F3 strains were selected on medium without tryptophan, the trp-4 gene is leaky; therefore, selection may not have been completely restrictive. To ensure that the compatibility was due to a novel suppressor and not to tol, the A/a compatible F3 strains from each of the seven crosses were tested for trp-4. None of the compatible F3 strains was tryptophan-requiring. All of the A/a incompatible F3 were tested for trp-4 to ensure that they were correctly scored as incompatible 48 because they had no suppressor, and not because they grew poorly due to the trp-4 gene. None of the incompatible F3 strains was tryptophan-requiring. Finally, to ensure that the compatibility was due to a suppressor and not to a novel gene which increased the rate of escape, 4 hyphal tips were obtained from 2 A/a compatible F3 strains from each of the seven crosses and were tested for mating type. If the "compatibility" were due to early escape, then the tips would be expected to be A or a, but not both; if the compatibility were due to a suppressor, then the tips would be expected to be A and a. The results are shown in Table 5. Almost all of the tips were A/a, suggesting that the compatible phenotype was due to a suppressor. Single mating type hyphal tips (all A) were found only in two of the suppressor strains, both derived from the same A/a escaped FI strain. When this A/a escaped FI strain escaped, a second mutation, in addition to the suppressor may have occurred. The second mutation could be one that causes instability of duplications. The single mating type mitotic segregants could have arisen by deletion of a during somatic growth or mitotic crossover between the L.G. I centromere and mt and between mt and ser-3 (only A products would have survived). Table 6 summarizes the findings described in Results 1. 49 Table 5 Mating Types of Hyphal Tips of A/a Compatible F3 Strains COMPATIBLE F3 TIP DERIVED FROM STRAIN: MATING TYPES OF TIPS a1-113-8 8 A/a a1-107-9 8 A/a a1-107-11 8 A/a A2-86-12 8 A/a A2-155-2 8 A/a A2-155-3 4 A/a, 4 A A2-155-5 6 A/a, 2 A Most of the hyphal tips were A/a, suggesting that the suppressors do not function by increasing the rate of escape. Table 6 Summary of Results 1 A/a ESCAPED F1 # SPORES FROM # TEMPERATURE- TEMPERATURE- # SPORES FROM # SPORES SECOND CROSS SENSITIVE SENSITIVE F2 THIRD CROSS COMPATIBLE 1-29 9 a1-107-9 37 19 1-54 27 / a1-107-11 40 28 1-58 16 1-59 24 / a1-113-7 31 0 1-65 19 / . a1-113-8 46 24 1-73 19 / a1-113-9 6 0 1-75 8 / a1-113-10 46 0 1-104 17 / y / 1-107 1 1 2 '/ 1-113 17 A ' a1-128-22 32 0 1-128 27 6 . a1-128-23 32 0 2-18 27 -—^ a1-128-24 25 0 2-20 12 a1-128-25 35 0 2-32 21 a1-128-26 32 0 2-37 1 1 a1-128-27 0 0 2-57 2 2-79 0 A2-86-1 30 0 2-86 20 3 ' A2-86-4 36 0 2-95 0 A2-86-12 30 9 2-103 31 2-110 0 2-117 37 A2-155-2 2-146 10 25 7 2-155 32 3 , A2-155-3 65 14 2-162 33 A2-155-5 46 18 KEY FOR A/a ESCAPED F1 1 or 2 - crossed to R601 or R602 in the first cross second # • isolate # from first cross KEY FOR TEMPERATURE-SENSITIVE F2 A or a - crossed to fl,A or fl,a in the second cross 1 or 2 • crossed to R601 or R602 in the first cross middle # • isolate # from first cross last # • isolate # from second cross 51 DISCUSSION 1 Seven strains containing novel suppressors of A/a incompatibility have been isolated. The strains were derived from 4 strains that had undergone escape events (strains #1-107, 1-113, 2-86 and 2-155), suggesting that there may be 4 novel suppressor mutations. None of the suppressors is allelic with tol or with the mating type gene. The seven strains produced A/a compatible progeny when crossed with a duplication-generating translocation strain. The A/a compatible progeny were probably duplications, not heterokaryons, because hyphal tips isolated from these strains, in general, contained both mating types. The compatible phenotype observed in these A/a progeny is due to new mutations that suppress A/a incompatibility, not to new mutations that increase the rate of escape from A/a incompatibility by increasing the rate of deletion or mitotic crossing over. There were surprisingly few temperature-sensitive F2 progeny from the 50 crosses of the second cross (18 of 329 tested). Two types of crosses produced no temperature-sensitive F2 progeny: fl, A x A/a escaped FI strains derived from R601 (un-3, A) and fl, a x A/a escaped Fl strains derived from R602 (un-3, a). In these 2 types of crosses, L.G. I from the fl parent had the same mating type gene as L.G. I from the temperature-sensitive parent; so perhaps the L.G. I homologs paired with the translocated segment which 52 has the opposite mating type (Fig. 10). When such pairings occur, fewer temperature sensitive progeny are produced. The L.G. I homologs probably did not pair with each other. If they did, temperature-sensitive progeny would have been produced. The alternative pairing hypothesis explains the absence of temperature-sensitive progeny from the two aforementioned types of crosses. When L.G. I from the fl parent pairs with the translocated DNA, no temperature sensitive progeny are produced. If there is a single crossover in the paired region, acentric and dicentric chromosomes are produced, and these, presumably, do not survive. If there is a double crossover in the paired region, the un-3 allele always segregates with an un-3+ allele. When L.G. I from the temperature-sensitive parent pairs with the translocated DNA, very few temperature sensitive progeny are produced. If there is a single crossover in the paired region, acentric and dicentric chromosomes are produced. If there is a double crossover in the paired region, only one of the 6 possible types of double crossover—one crossover on each side of the mating type gene—gives temperature sensitive progeny. This type of double crossover is rare because of the proximity of un-3 to the mating type gene. If one disregards the two types of crosses that yielded no temperature-sensitive F2 progeny, the proportion of temperature-sensitive F2 strains is still low (18 of 165). In the two types of crosses that did give temperature-53 PAIRING SEGREGATION ser un + a ser* un+ A ser+ un la lb Ha, la lib, lb ser un+ a ser* un A ser* un+ A lb la lla, lb lib, la One explanation for the low number of temperature-sensitive F2 strains is the alternative pairing hypothesis. Either of the L.G.I homologs could have paired with the translocation. r_ tne L.u.i nomoiogs couia nave pairea witn im nlyUre lU Thin vertical lines indicate regions of pairing 54 sensitive F2 progeny (R601-derived escaped strains x fl, a and R602-derived escaped strains x fl, A), perhaps pairing of the L.G. I homologs with each other, as depicted in Fig. 7, did not always occur. If either of the L.G. I homologs had paired with the translocated DNA, fewer temperature-sensitive progeny would have been produced. Two additional explanations for the low number of temperature-sensitive progeny are possible. Duplication strains undergo a process called RIP ("Repeat-Induced Point mutation", previously "Rearrangements Induced Premeiotically") during mating, with the result that duplicated genes are often mutated (Selker and Stevens, 1985; Selker, et al., 1987; Selker and Garrett, 1988; Cambareri, et al., 1989; Grayburn and Selker, 1989; Selker, 1990; Cambareri, et al., 1991). RIP of either un-3 or un-3+ could have produced un-3 nulls, which are lethal (Lambowitz, unpublished results, Glass, personal communication). Finally, germination of un-3 spores may occur less frequently than germination of wild type spores (Glass and L. Stenberg, personal communication), possibly because of the heat shock required to initiate germination. A/a duplication strains escaped from incompatibility by several different means other than the generation of suppressors. The 8 phenotypic classes of escaped Fl strains, shown in Fig. 6, could have been the results of deletion/mutation at the mating type locus, RIP, mitotic 55 crossing over or low fertility due to the presence of duplicated genetic material. Any of the 8 phenotypic classes of escaped Fl strains could have arisen by deletion/mutation at one or both of the mating type loci. Experiments performed by DeLange and Griffiths (1975) on escape from A/a incompatibility suggested that mixed mating type heterokaryons often escaped by deletion of one mating type. Similar events could have occurred in mixed mating type duplication strains. The 8 classes of escaped strains could have arisen by RIP. A/a duplications are not really duplications because A and a are idiomorphs, not alleles (Metzenberg and Glass, 1990), i.e. the central portions of the DNAs are completely different from each other (Glass, et al., 1988). The flanking regions, however are virtually identical (Glass, et al., 1988) and RIP can sometimes occur in unique sequences close to the duplicated ones (Foss, et al., 1991). The single mating type escaped strains, classes 7 and 8, could have been generated by mitotic crossing over. Once the mating type genes had segregated into different nuclei, one type of nucleus could have outgrown the other and the resultant culture would only have had a single mating type reaction. The barren phenotype associated with duplications in N. crassa could account for classes 2-6. Duplication strains sometimes produce abundant perithecia, but very few 56 ascospores (Newmeyer and Taylor, 1967). If this occurred, the spores simply may have been overlooked during scoring. What is tol? It is not possible to formulate a comprehensive statement regarding the biological significance of incompatibility in all of the fungi, for it manifests itself in many different forms. Incompatibility can be heterogenic, preventing fusion between strains of different genotypes, either vegetative fusion (e.g. in W. crassa), sexual fusion (e.g. in Sordaria fimicola (Olive, 1956) and Ceratostomella radicicola (El-Ani, et al., 1957)) or both vegetative and sexual fusion (e.g. in Aspergillus nidulans (Jinks, et al., 1961) and Podospora anserina (Esser, 1971)); or it can be homogenic, preventing fusion between strains of similar genotypes, either sexual fusion (e.g. in W. crassa) or both vegetative and sexual fusion (e.g. in Schizophyllum commune (Raper, 1966 cited in Burnett, 1975)). The significance of each incompatibility system, because of the diversity, must be considered separately. Vegetative incompatibility controlled by the mating type genes of N. crassa may be biologically unrelated to incompatibility controlled by the het genes. This idea is supported by the existence of the gene, tol, which suppresses A/a incompatibility without affecting het-C/c or het-JE?/e incompatibility (Newmeyer, 1970; Perkins, 1974). 57 Vegetative incompatibility seems to be an intrinsic function of the mating type genes. Genetic and molecular data support this idea and suggest that compatibility mechanisms evolved secondarily. Metzenberg and Ahlgren (1973) introgressed the mating type genes of N. tetrasperma into a largely N. crassa background. The resultant strains demonstrated incompatibility in mixed mating type heterokaryons and in heterozygous duplications, suggesting that the W. tetrasperma genes had the ability to instigate the incompatibility reaction. In their own environment, in N. tetrasperma, the mating type genes must either be suppressed for the incompatibility function or lack target genes or both. A recent study by D. Jacobson (personal communication) suggests that one way in which N. tetrasperma tolerates A/a ascospores is by the presence of the suppressor, tol. He introgressed sequences of N. tetrasperma corresponding to tol of W. crassa into a N. crassa background. The resultant strain of N. crassa behaved as if it were tol, not tol+. The mating type genes of N. sitophila, like W. tetrasperma, are able to produce the incompatibility phenotype in a W. crassa background (Perkins, 1977). N. sitophila mating type genes were introgressed into W. crassa, whereupon the N. sitophila genes exhibited incompatibility in A/a duplications. Even more interesting was the observation that the suppressor gene, tol, eliminated the incompatibility, indicating that tol is 58 unable to detect any significant difference between W. crassa and N. sitophila mating type genes. A result with similar implications was obtained by Glass (personal communication). Replacement of the N. crassa A gene with the N. africana A gene did not alter the ability of the transformant to initiate the incompatibility reaction, again implying that the mating type gene from a homothallic species is capable of orchestrating vegetative incompatibi1ity. Incompatibility controlled by the vegetative incompatibility genes may have developed independently of A/a incompatibility. There are many theories regarding the biological benefits of heterokaryon incompatibility. Caten (1972) suggested that incompatibility systems exist to limit the spread of infectious viruses or cytoplasmic determinants. Later studies indicated, however, that plasmids can cross incompatibility barriers within a species (Collins and Saville, 1990) and even between species (Griffiths, et al., 1990), presumably during brief periods of unstable fusion. Hartl, et al. (1975) suggested that incompatibility prevents the exploitation of an adapted mycelium by a less well adapted one growing in the same niche. Although this hypothesis could apply to a fungus like N. crassa, in which a defective homokaryon can survive by fusion with another mycelium, it does not seem a likely explanation for 59 incompatibility in fungi like the basidiomycetes in which fusion leads to mating. It has been suggested that incompatibility serves to distinguish individuals, which may be important in the maintenance of fine-tuning between nuclei and organelles. Considering that nuclei are associated with different organelles every time mating occurs, this does not seem to be a likely explanation for the existence of incompatibility. J. Begueret (personal communication) succeeded in creating a novel incompatibility group via mutation in Podospora, which led him to propose that incompatibility genes are nothing more than mutations without biological significance. Jinks, et al. (1961) made a similar suggestion in their study of incompatibility in Aspergillus nidulans. They maintained that heterokaryon incompatibility is a consequence of genetic diversity, not a cause. This idea would only apply to fungi like N. crassa in which the incompatibility reaction does not affect mating. If a novel allele arose in a population, it would be selected against because the cell containing it would die if it fused with an unlike type. A novel allele would not be selected against if it arose in a spore that established a separate population before encountering incompatible fungal types. In the basidiomycetes, however, a novel incompatibility group would be able to mate with all other existing groups, allowing it to spread quickly through a population. 60 The gene, tol+, and the new suppressors could be mating type target genes, controlling different steps in the pathway to A/a incompatibility, and when they are mutated, the reaction fails to occur. Their relationships to each other in terms of where they fit in to the pathway are unknown at present. They could be mutants in sequential reactions or reactions that occur simultaneously. The suppressors could be enzymes required for vegetative growth, normally turned off by the A/a product (directly or indirectly) during incompatibility. The mutants could be altered such that they are no longer recognized by the regulating product and are, therefore, not repressed and the incompatibility reaction does not occur. The suppressors could be required for recognition of vegetatively growing A or a hyphae. The mutants, in this case, could be defective for vegetative recognition, thus eliminating the incompatibility reaction. The suppressors could produce toxic metabolites in the presence of the A/a product. These mutants could be defective for toxin production itself or for regulation so that the incompatibility reaction does not occur. The existence of other genes affecting the incompatibility reaction raises a question regarding N. tetrasperma. As previously mentioned, one strain of N. tetrasperma appears to contain the recessive allele of tol (Jacobson, personal communication). What is the state of the other suppressor genes? As long as a strain has tol, it 61 will be heterokaryon compatible, so the alleles of the other suppressors would not affect the phenotype. Until more is known about the relative functions of the new suppressors, it is not possible to predict the suppressor genotype(s) of W. tetrasperma. Perhaps there are suppressors that, unlike tol, suppress both A/a incompatibility and mating. If so, it would mean that mating and A/a incompatibility act through at least one common step. Perhaps there are A/a incompatibility suppressors that also suppress incompatibility controlled by one or some of the net genes. If so, it would imply that incompatibility is effected through one pathway and that tol acts before or after the common part(s) of the pathway. The number of different suppressor genes may give an indication of the number of steps involved in generating the incompatibility reaction. 62 INTRODUCTION 2 A molecular picture of the mating type genes is emerging with the aid of mating type mutants (Griffiths and DeLange, 1978; Griffiths, 1982; Griffiths, personal communication) and molecular biological techniques (Glass, et al., 1988; Staben and Yanofsky, 1990; Glass, et al., 1990). The A idiomorph, as defined by its region of non-homology with a, is 5301 base pairs in length (Glass, et al., 1990), and a, by the same definition, is 3235 base pairs in length (Staben and Yanofsky, 1990) (Fig. 11). All of the Am mutants (Griffiths, 1982) that have been sequenced map within the single ORF, called A-l. All of the Am and am mutants of Griffiths and DeLange (1978) that have been sequenced map within the exons in the ORFs (Glass, et al., 1990; Staben and Yanofsky, 1990). Some examples follow. One of the mutants, anl, has a frameshift due to the deletion of a single base pair. The insertion of 212 base pairs, which characterizes the mutant am30 causes a premature transcription stop. The unique compatible, fertile mutant, am33, has a base pair substitution which is located farther downstream than either of the other two mutations. Species in other genera have idiomorphs instead of alleles at their mating type loci, e.g. Saccharomyces LEGEND Thick lines • identical flanking sequences Filled boxes - idiomorphs Open boxes • postulated ORFs Arrows - presumed transcripts with introns Mating type regions of N. crassa (from Staben and Yanofsky, 1990). 64 cerevisiae (Strathern, et al., 1980; Nasmyth and Tatchell, 1980), Schizosaccharomyces pombe (Kelly, et al., 1988), Ustilago maydis a gene (M. Bolker and R. Kahmann, 1991, in published abstracts from Sixteenth Fungal Genetics Conference) and Podospora anserina (Coppin, 1991, in published abstracts from Sixteenth Fungal Genetics Conference). The mating type genes of N. crassa are incompatible not only in a heterokaryon, but also in one nucleus. Newmeyer (1970) used nuclear incompatibility to induce tol, which suppresses both types of incompatibility. Glass, et al. (1990) observed a 100-fold reduction in transformation efficiency of the ORF, A-l, into a spheroplasts, compared to the transformation efficiency into A spheroplasts; and Glass (personal communication) observed a similar reduction of transformation efficiency of a sequences into A. Staben and Yanofsky (1990) also reported a decrease in the frequency of A transformants when the donor DNA was ORF, a-l, as compared to when the donor DNA was a portion of the a idiomorph not including a-l. Nuclear incompatibility may be separable from heterokaryon incompatibility. Glass and Griffiths (personal communication) created a A-duplication strain by transforming a strain of mating type A with the ORF of the sterile, compatible mutant, Am64. In order to RIP the A gene, they crossed the transformant to a strain of mating type a. Surprisingly, some of the progeny displayed the 65 incompatibility phenotype. Growth occurred as a small wispy knot of mycelia with no aerial hyphae. All of the cultures escaped from slow growth within one week, and then showed a mating type a reaction when tested. If the ORF had segregated from mt as an independent locus, then one half of the a spores would have contained Am64 and it is possible that these were the incompatible strains (Fig. 12). If the incompatible strains were Am64/a, it appears as though Am64 has lost heterokaryon incompatibility while retaining nuclear incompatibility. At first it seemed possible that the incompatible phenotype was due to residual heterokaryon incompatibility specified by Am64, but this hypothesis has been discarded because a mixed mating type heterokaryon of An64 grew as fast as a positive control strain. To test the possibility that mutations at mt can eliminate heterokaryon incompatibility without eliminating nuclear incompatibility, duplication strains that contained the fertile, compatible mutant, am33, and A were examined for their morphological characteristics and growth rates. A/am33 duplication strains were made by crossing 3 different am33-containing strains to A strains containing a translocation of the mating type gene. The mutant, am33, is known to be compatible in a heterokaryon with A, although the growth rate of an am33 + A heterokaryon has not been measured precisely before now. It was measured to determine if am33 na<j ariy residual heterokaryon incompatibility. 66 1/2 Am64 1/2 A NORMAL OR RIP 1/2 Am64 INCOMPATIBLE? 1/2 a / \ NORMAL a re 12 Segregation of Am64 ORF and resulting phenotypes. 67 RESULTS 2 The growth rate of the sterile, compatible mutant, Am64f ^n a mixec| mating type heterokaryon, Am64 + 7 (ad-3B, a), was measured to determine if Am64 had residual incompatibility. The growth rate was compared to that of the component strains alone and to that of an incompatible mixed mating type heterokaryon, 153 (ad-3A, nic-2, A) + 7 (ad-3B, a). The heterokaryon, Am64 + 7 (ad-3B, a), grew as fast as the component strain 7 (ad-3B, a), and faster than the incompatible mixed mating type heterokaryon, 153 (ad-3A, nic-2, A) + 7 (ad-3B, a) (Table 7 and Fig. 13), suggesting that the mutant, Am64, has lost its heterokaryon incompatibility function completely . The following section describes a series of tests done to study heterokaryon and nuclear incompatibility in another mating type mutant, am33. The growth rate of am33 in a mixed mating type heterokaryon (am33, ad-3B + 1-22-83 (ad-3A, nic-2, un-3, A)) was measured to determine if the mating type mutant had residual incompatibility. The growth rate was compared to several controls: (1) an incompatible mixed mating type heterokaryon (51-2 (ad-3B, cyh-1, a) + 1-22-83 (ad-3A, nic-2, un-3, A)); (2) a mating type homokaryon—am33, ad-3B(128) paired with 51-2, a strain of mating type a (ad-3B(114), cyh-1, a); (3) a compatible mixed mating type heterokaryon in which both components had tol (1-9-57 68 Table 7 Strains and Media Used in the Measurement of Growth Rate of Am6 in a Mixed Mating Type Heterokaryon GENOTYPE STRAIN mt AUXOTROPHIC GENES OTHER 153 * A ad-3A, nic-2 un-3, cyh-1 7 a ad-3B Am64 Am64 ad-3A, nic-2 un-3, cyh-1 MINIMAL MEDIUM + POSITIVE CONTROLS Am64 adenine, nicotinic acid 7 adenine NEGATIVE CONTROLS Am64 7 153 + 7 EXPERIMENTAL Am64 + 7 ad-3A - adenine-requiring (complements ad-3B) ad-3B - adenine-requiring (complements ad-3A) nic-2 • nicotinic acid- or nicotinamide-requiring un-3 • temperature-sensitive cyh-1 « cycloheximide-resistant * 153 was the strain used to generate Am64. 69 24 Jl 120 168 216 264 312 380 48 96 144 192 240 288 336 384 fme (hois) Growth rate of Am64 in a mixed mating type heterokaryon. Piniiro 1Q Each line represents the average of a minimum of 3 ilyUr© lO measurements of growth rate. 70 (ad-3B(128), tol, a)+ 1-9-3 (ad-3B(114), tol, A)); and (4) the following component strains on supplemented media: 1-22-83; am33, ad-3B-, and 51-2. The heterokaryon, am33 + 1-22-83, grew as fast as the mating type homokaryon, am33 + 51-2 and the compatible mixed mating type heterokaryon, 1-9-57 + 1-9-3; and faster than the incompatible heterokaryon, 51-2 + 1-22-83 (Table 8 and Fig. 14), suggesting that the mutant, am33, has lost its heterokaryon incompatibility function completely. To determine the phenotype shown by am33 in the same nucleus with A, duplication progeny were obtained from the following crosses. Three am35-containing strains, am33, ad; Rl-14 and Rl-29 (Table 9) were crossed to the translocation strain, T(I->II) 39311, ser-3, A, to generate duplication progeny. The crosses are diagrammed in Figs. 15A and 15B. Single spores were viewed through a dissecting microscope and collected with a tungsten needle. Individual spores were placed into slants of supplemented medium (minimal medium + adenine for the am33, ad-derived spores; and minimal medium + nicotinic acid and pantothenic acid for the Rl-14- and Rl-29-derived spores). The test tubes were placed into a 60°C water bath for 30 minutes to initiate germination of the spores, which were examined after 3 days. The progeny from each of the 3 crosses expressed one of two growth phenotypes. One phenotypic class, called "inhibited", grew slightly less vigorously than the other, "healthy". The inhibited phenotype was distinct from the 71 Table 8 Strains and Media Used in the Measurement of Growth Rate of am33 in a Mixed Mating Type Heterokaryon STRAIN GENOTYPE 1-22-83 A, ad-3A, nic-2, un-3 51-2 a, ad-3B(114), cyh-1 I-9-57 a, ad-3B(128), tol I-9-3 A, ad-3B(114), tol MINIMAL MEDIUM + POSITIVE CONTROLS 1-9-57 «• 1-9-3* — am33, ad + 51-2 — I-9-3 adenine am33, ad adenine 51-2 adenine I-22-83 adenine, nicotinic acid I-9-57 adenine NEGATIVE CONTROLS 51-2 + I-22-83** — I-9-3 — am33, ad — 51-2 — 1-22-83 — 1-9-57 — EXPERIMENTAL am33, ad + 1-22-83 — ad-3A - adenine-requiring ad-3B(114) - adenine-requiring ad-3B(128) - adenine-requiring un-3 • temperature-sensitive cyh-1 • cycloheximide-resistant * The alleles, ad-3B(114) and ad-3B(128), complement. ** The genes, ad-3A and ad-3B, complement. 72 LEGEND 1 • I-9-57 + I-9-3 2 • am33,ad + 51-2 3 - 51-2 + 1-22-83 4 - am33,ad + 1-22-83 Growth rate of am33 in a mixed mating type heterokaryon. r- llKn *A Each line represents the average of 2 measurements Figure I*f of growth rate. 73 Table 9 Genotypes of am33 Strains STRAIN GENOTYPE SOURCE am33, ad am33, ad-3B (128) AJ.F.G. R1-14 am33, nic-3, pan-1, al-1 (#43) N.L.G. R1-29 am33, nic-3, pan-1, al-1 (#29) N.L.G. am33 - fertile compatible mating type mutant ad-3B (128) - adenine-requiring (allele #128) nic-3 - nicotinic acid- or nicotinamide-requiring pan-1 - pantothenic-acid requiring al-1 - albino #43 - isolate # of N.L.G. #29 - isolate # of N.L.G. A.J.F.G. - A.J.F. Griffiths N.L.G. - N.L. Glass 74 am33, ad-3B x T(l->ll)39311, ser-3, A am33 ad | 32o/0 am33 ad | 32% •M-18% am33 18% ad | ser A minimal mediLm + adenine 50% 50% 50% 50% 50% 50% 50% 50% ser A ser A ser A ser A II II II II am33, ad A/am33, ad (incompatible?) dead dead am33 A/am33 (incompatible?) dead dead Cross made to generate A/am33 duplications. Genetic r:_liro jr A distance between ad-3B and mt is 36% rigUre IOH (Perkins, et al., 1982). 75 R1-14 or R1-29 x T(l->ll)39311, ser-3, A am33 al-1 -x-pan-1 IV ser-3 A nic-3 VII pantothenic acid + nicotinic acid am33 50% I 50% 50% II II ser-3 A am33 A/am33 (incompatible?) 50% H 1 50% 50% dead dead ser-3 A Figure 15B Crosses done to generate A/am33 duplications. 76 standard incompatibility phenotype (dark agar) in that the growth was more luxuriant. Mating type tests were done on all of the isolates (Table 10). If perithecia were produced with the fl tester, the reaction was scored as positive. Crosses, therefore, could have been barren. The healthy progeny from all three crosses were either a, which was expected (Fig. 15A and 15B) or A/a, which was unexpected. The inhibited progeny were mostly A/a, which was the result predicted in the hypothesis, although the predicted phenotype was dark agar. One inhibited isolate could have contained both mating types, but the a mating type reaction was difficult to confirm. One inhibited isolate reacted as only a. The significance of the four types of progeny is considered in Discussion 2. In order to verify the difference in phenotypes, sample progeny from each cross were inoculated onto plates. The difference between the phenotypes became more evident under these growth conditions. As a control, a A/a, tol duplication strain, which has a phenotype known as "square agar" (Newmeyer, 1970), very slightly different from wild type, was also inoculated onto a plate. All of the healthy progeny tested, including the single and double mating type isolates, and the square agar control grew to cover the plate with an even layer of mycelia. Most of the inhibited progeny (22 of 30) grew in a dense mat in the centre of the plate with a few hyphae extending beyond 77 Table 10 Mating Types of Progeny CROSS SPORE TYPE MATING TYPE am33,ad-3B x T(l->ll)39311,ser,A 17 h 12 A/a 5 a 12 i 11 A/a 1 A/? R1-14 x T(l->ll)39311,ser,A 9 h 6 A/a 3 a 11 i 11 A/a R1-29 x T(l->ll)39311,ser,A 7 h 3 A/a 4 a 7 i 6 A/a 1 a h • healthy (i.e. vigorous growth) i • inhibited (i.e. less vigorous growth) The A/a healthy isolates were unexpected. 78 the central mat (Table 11). Some of the progeny originally scored as inhibited (8 of 30) grew evenly on plates. Since the plating test was done several months after the initial spore isolation, it is possible that these progeny had escaped. Strains were kept in the freezer (-20°C) during most of this time, which could explain why all of the inhibited progeny had not escaped. The growth rates of all of the isolates were measured and compared to incompatible duplication (A/a,tol+) and compatible duplication (A/a,tol) controls. Fungi were inoculated at one end of a 50 cm growth tube containing 30 mL of supplemented medium for the isolates and minimal medium for the controls. The mycelial fronts were marked at regular intervals. Results are shown in Figs. 16-29. The slopes of the graphs are shown in Table 12. All of the healthy isolates grew at the same rate as their siblings, as fast as the compatible controls, and considerably faster than the incompatible controls. The growth rates of the inhibited strains varied widely. Only one strain, the single mating type inhibited strain, 29-i-7, grew as fast as the compatible controls and healthy strains. The rest grew at rates intermediate between those of the compatible and incompatible controls. The significance of these results is considered in Discussion 2. The following test was done to determine whether the mating types of the A/a strains had segregated into different nuclei. Conidial samples from the crosses with 79 Table 11 Phenotypes of Progeny STRAIN # OF ISOLATES WITH PHENOTYPE OF DENSE MAT EVEN GROWTH a-i-x 8 4 a-h-x 0 17 14-i-x 10 1 14-h-x 0 9 29-l-x 4 3 29-h-x 0 7 KEY a, 14 or 29 • strains am33,ad; R1-14 or R1-29 h or i = healthy or inhibited x s isolate # Inhibited strains with even growth were probably escapes. 80 the females, Rl-14 and Rl-29, were plated on sorbose plates containing pantothenic acid and nicotinic acid. Single conidial colonies were cut out of the agar, grown and tested for their mating types. All of the inhibited isolates tested contained both mating types, suggesting that the mating types had not segregated mitotically. The healthy isolates of strains which were originally A/a were all a (Table 13). The significance of these results is discussed below. 81 Growth Rates of Controls 45 0 48 96 144 192 24 72 120 168 Tmejhotrs) LEGEND FOR FIGURES 16-29 a, 14 or 29 - strains am33, ad; R1-14 or R1-29 h or i - healthy or inhibited x - isolate # Figure 16 82 Figure 17 83 Figure 18 84 Figure 19 85 Growth Rates of a-i-x 0 24- 48 72 96 120 144 168 192 Trrefws) Figure 20 86 Growth Rates of a-i-x Figure 21 87 Growth Rates of 14-h-x SOi 0 24 € 72 96 Tmefuis) Figure 22 88 Figure 23 89 Growth Rates of 14—i—x 0 24 48 72 96 120 144 168 192 rine (hours) Figure 24 90 Growth Rates of 0 24 48 72 96 120 144 168 192 rme (heirs) Figure 25 91 Figure 26 92 Figure 27 93 Growth Rates of 2H-x 0 24 48 72 96 120 144 1® 192 fire (bars) Figure 28 94 Growth Rates of 29-i-x Figure 29 95 Table 12 Slopes of Growth Rates of Progeny STRAIN SLOPE-A/a, tol+ (average of 2 strains) A/a, tol (average of 2 strains) a-h-x (average of 17 strains) a-i-1 a-i-2 a-i-3 a-i-4 a-i-5 a-i-6 a-i-7 a-i-8 a-i-9 a-i-10 a-i-11 a-i-12 14-h-x (average of 9 strains) 14-i-x (average of 14-i-1, 2, 3, 6, 7) 14-i-x (average of 14-i-4, 5, 9) 14-i-8 14-i-10 14-i-11 29-h-x (average of 7 strains) 29 29 29-i 29 29 29 29 -1 -2 -3 -4 -5 -6 -7 ,1 1 .17 .0035 .50 .50 .27 .33 .48 .42, .44 .12 .47 .23 .38, .46 .31 .34 .51 .39 .056 .096 .49 .044, .26 .50 .19 .43 .053 .37 .34 .13 .52 * Slopes were measured at straight regions of the graph, over no fewer than 72 hours (except for the first slope for a-i-9, which was measured over 48 hours). All except 1 (29-i-7) of the inhibited strains grew more slowly than the healthy strains and the positive control strains (A/a, tol). 96 Table 13 Mating Types of Single Conidial Isolates of Progeny STRAIN MATING TYPE 14-h-4-1 a 14-h-4-2 a 14-h-4-3 a 14-h-5-1 a 14-h-5-2 a 14-h-5-3 a 14-h-6-1 a 14-1-3-1 A/a 29-h-5-1 a 29-h-5-2 a 29-h-6-1 a 29-h-6-2 a 29-h-6-3 a 29-h-7-1 a 29-h-7-2 a 29-h-7-3 a 29-1-1-1 A/a 29-J-1-2 A/a 29-J-1-3 A/a 29-1-3-1 A/a 14 or 29 - strain R1-14 or R1-29 h or i • healthy or inhibited first # - spore isolate # last # - conidial isolate # Healthy strains were a; inhibited strains were A/a. 97 DISCUSSION 2 The findings of Results 2 present evidence to support the idea that at least 2 mating type mutants completely deficient in heterokaryon incompatibility, Am64 and am33, retain nuclear incompatibility. The growth rates of the two mutants in mixed mating type heterokaryons equalled those of positive controls and surpassed those of negative controls, so it appears that neither mutant has residual heterokaryon incompatibility. The nuclear incompatibility seen, therefore, was not due to residual heterokaryon incompatibi1ity. Four phenotypes were seen in the progeny from the three crosses made to generate A/am33 duplications: (1) single mating type healthy (12 isolates), (2) double mating type healthy (21 isolates), (3) single mating type inhibited (1 isolate) and (4) double mating type inhibited (28, or possibly 29, isolates). The only phenotype that was expected (Fig. 15A and 15B) was the first phenotypic class, single mating type healthy, which were normal segregants from the cross. The phenotype of the duplicated progeny was unknown, although it was predicted to be dark agar. The fourth phenotypic class, the double mating type inhibited strains, is the one providing evidence that nuclear incompatibility can exist in the absence of heterokaryon incompatibility. All of the A/a inhibited 98 strains grew more slowly than the healthy strains. The reduced growth rates could have been due to the presence of am33 and A in the same nucleus. Four of the seven A/a inhibited strains that grew evenly on plates (a-i-3, a-i-7, 14-i-lO, 29-i-2) had the highest growth rates of all of the A/a inhibited strains, supporting the idea that they had escaped and were growing at rates higher than the inhibited strains, yet lower than the healthy strains. Two of the seven strains (a-i-4, a-i-9) had growth rates that were high at first, but suddenly dropped. One of the seven strains (29-i-6) had an irregular growth pattern. The growth rates of the A/a inhibited strains were highly variable. The variant rates could be characteristic of nuclear incompatibility itself. They could also have been produced by escape, either by a number of different mechanisms or by the same mechanism producing different results. Perhaps the inhibited strains showed various growth rates because they were in different stages of escape by somatic segregation and overgrowth by a nuclei. The second phenotypic class, the A/a healthy isolates was not expected. Metzenberg (personal communication) suggested that these strains grew vigorously because the two mating types had segregated somatically into separate nuclei producing A + am33 heterokaryons, which are known to be compatible. The translocation is long enough to sustain a double crossover. To test his hypothesis, single conidial isolates were obtained from healthy and inhibited strains 99 and tested for their mating types. If Metzenberg's hypothesis were correct, conidia derived from healthy strains would have been A or a, whereas conidia derived from inhibited strains would have been A/a. These results were observed, with the exception that there were no A conidial isolates from the healthy strains. If the healthy phenotype were due to mitotic segregation, the cultures would have contained both mating types shortly after germination of the spores. After a period of time, it is conceivable that the a nuclei overgrew the A nuclei because the latter contained the ser-3 marker which is closely linked to the mating type gene. Overgrowth by a nuclei may have been slowed by cross-feeding of the serine-requiring A nuclei by the a nuclei. The healthy strains grew as fast as the compatible controls (Figs. 16-19, 22, 23, 26 and 27). The compatible controls were A/a duplications containing tol. The evidence presented above suggests that the healthy strains were simply a*33 strains. The third phenotypic class, the 1 unexpected inhibited strain that reacted with only one mating type (29-i-7), could have escaped from the inhibition by deletion of A. It grew evenly on a plate, and had a growth rate that exceeded some of the healthy strains, suggesting that it had escaped from inhibited growth. An explanation is needed for why some A/am33 strains (the healthy ones) escaped early, while others (the 100 inhibited ones) escaped later or not at all. Would all of the inhibited strains have escaped eventually? Was the time of escape the only difference between the healthy and inhibited strains or was there a difference in the mechanisms of escape? Was it significant that escaped inhibited strains did not grow as fast as healthy strains? Was there a gene segregating that caused early/late escape by mitotic crossing over in the A/a healthy strains and the A/a inhibited strains? The first of the three crosses segregated half A/a healthy and half A/a inhibited progeny. In the other 2 crosses, the sample sizes were probably too small to reflect accurate ratios. These questions can only be addressed by further study. Perkins (personal communication) has observed that A/am33 duplications grow with an abnormal morphology which he calls "square" because Newmeyer (1970) called the morphology associated with A/a, tol duplications "square". Perkins' observations are not inconsistent with those presented above, except that Newmeyer's square strains grow at wild type rates, whereas Perkins' square strains, assuming they exhibit the same growth rates as those discussed here, grow at sub-wild type rates. For this reason, A/am33 duplications should be referred to by some name other than square. 101 Molecular Model One current model of the molecular interactions that occur during mating is as follows (Metzenberg and Glass, 1990r Glass, personal communication). A combination product of A and a effects incompatibility during the vegetative cycle, when the mating type genes are expressed at low levels, and the same product instigates mating functions during the sexual cycle, when the mating type genes are expressed at higher levels. Nuclear incompatibility can be explained in the context of this model. There is obviously a difference in the function of the mating type products during the vegetative and sexual cycles. Perhaps the mutant, Am64, is defective for mating, but not incompatibility, as previously believed. It, and the other mutant studied here, am33, could be defective in terms of the stability of their products. Both mutants are functional for nuclear incompatibility and defective for heterokaryon incompatibility. In terms of the stability hypothesis, heterokaryon incompatibility requires more stable mating type products than nuclear incompatibility. These results can be explained as follows. 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