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Mating-type-associated vegetative incompatibility in Neurospora crassa Shiu, Patrick Ka Tai 2000

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MATING-TYPE-ASSOCIATED VEGETATIVE INCOMPATIBILITY  I N NEUROSPORA by  CRASSA ?  .  PATRICK K A TAI SHIU B . S c , University of British Columbia, 1994 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF  DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Biotechnology Laboratory and Department of Botany)  W e accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A July 2000 © Patrick K a Tai Shiu, 2000  In presenting degree  this  thesis  in  at the < University of  freely available for reference copying  of  department  British Columbia, and study.  by  his  of this  or  her  Department of  Botany  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  A u <  ?  1  5  '  2  the  0  0  0  requirements  I agree  I further agree  representatives.  may be It  thesis for financial gain shall not  permission.  Date  of  this,.thesis for scholarly purposes or  publication  partial fulfilment  is  that the that  an  advanced  Library shall make it  permission for extensive  granted by the understood  be  for  allowed  that without  head  of  my  copying  or  my written  Abstract t  The mating-type locus in Neurospora crassa controls mating and sexual development. The fusion of reproductive structures of opposite mating-type, A and a, is required to initiate sexual reproduction. However, the fusion of hyphae of opposite mating-type during vegetative growth results in growth inhibition and cell death, a process that is mediated by the tol locus. Mutations in tol are recessive and suppress mating-type associated heterokaryon incompatibility; heterokaryon incompatibility is a mechanism that prevents the formation of vigorous heterokaryons between genetically dissimilar individuals. In this study, the functional domain for heterokaryon incompatibility in the A mating-type protein, specifically M A T A - l , is mapped to a leucine-rich repeat. The tol gene encodes a putative 1011-amino acid polypeptide with a coiled-coil domain and a leucine-rich repeat (both essential for heterokaryon incompatibility). It contains a region of similarity with two het genes (HET-6 and HET-E). Repeat-induced point mutations in tol result in mutants that are wild-type during vegetative growth and sexual reproduction, but which allow opposite mating-type individuals to form a vigorous heterokaryon. Transcript analyses show that tol mRNA is present during vegetative growth but absent during a cross. These data suggest that tol transcription could be repressed in order to allow the co-existence of opposite mating-type nuclei during the sexual reproductive phase, tol is expressed in a mat A, mat a, A/a partial diploid and in a mating-type deletion strain, indicating that M A T A - l and M A T a-l are not absolutely required for transcription or repression of tol. These data suggest that T O L may interact with M A T A - l and/or M A T a-l to form a death-triggering complex. To understand how tol may mediate mating-type incompatibility, attempts have been made to isolate TOL-interacting proteins {top) by yeast 2-hybrid system. Five N. crassa mycelial cDNA-pGAL4 A D clones are shown to have plasmid-dependent interaction with T O L in yeast. One of them is a homologue of Schizosaccharomyces pombe vip-1, which is a p53related protein. The tol gene is apparently conserved in many different Neurospora species. One particular allele, tol from N. tetrasperma, is of special interest. tol is a natural tot in the regard 7  T  that it does not mediate mating-type incompatibility. Evidence provided in this thesis suggests that two loci closely linked to to? may mediate non-allelic vegetative incompatibility and sexual incompatibility, respectively.  Table of Contents Abstract List of Tables List of Figures Acknowledgements  ii vi vii viii  1. Overview and Introduction  1  1.1 T H E S I S O B J E C T I V E S 1.2 G E N E R A L I N T R O D U C T I O N T O NEUROSPORA  1 CRASSA  1  1.2.1 Neurospora crassa, a genetic model organism  2  1.2.2 Advantages of Neurospora  4  1.3 F U N G A L M A T I N G - T Y P E S  5  1.3.1 Saccharomyces cerevisiae Meyer ex Hausen (hemiascomycete; unicellular yeast)....6 1.3.2 Schizosaccharomycespombe Lindner (hemiascomycete; fission yeast)  12  1.3.3 Neurospora crassa Shear & Dodge (heterothallic pyrenomycete)  16  1.3.4 Podospora anserina (cas.) Niessl (pseudohomothallic pyrenomycete)  23  1.3.5 Cochiobolus heterostrophus (Drechsler) Drechsler (heterothallic loculoascomycete) 25 1.3.6 Sordaria macrospora Auersw. (homothallic pyrenomycete) 1.3.7 Ustilago maydis (DC.) Corda (heterobasidomycete)  27 28  1.3.8 Coprinus cinereus (SchaeffenFr.) Gray and Schizophyllum commune Fr. (homobasidiomycete) 1.3.9 Other filamentous ascomycetes 1.4 H E T E R O K A R Y O N I N C O M P A T I B I L I T Y I N F U N G I  36 38  1.4.1 Introduction to heterokaryon incompatibility  38  1.4.2 N. crassa mat locus  40  1.4.3 N. crassa het-c locus  42  1.4.4 N. crassa het-6 locus  46  1.4.5 P. anserina het-s locus  48  1.4.6 P. anserina het-c/e (het-c/d) system  2.  32  51  1.4.7 P. anserina idi genes (mduced during incompatibility)  54  1.4.8 Suppressor (modifier) genes for heterokaryon incompatibility i n P. anserina  56  Functional Analysis of matA-1 Gene of Neurospora crassa  60  2.1 I N T R O D U C T I O N  60  2.2 M A T E R I A L S A N D M E T H O D S  61  2.2.1 Strains, media and culturing  61  2.2.2 D N A isolation and P C R amplification  61  2.2.3 Plasmid construction for mat A-l and FMR1  62  2.2.4 Site-directed mutagenesis and chimeric construction  62  2.2.5 D N A sequence analyses  63  2.2.6 Transformation reduction assay for heterokaryon incompatibility  64  2.3 R E S U L T S  64  2.3.1 P C R amplification and construction of various mat A-l (A ) mutant constructs  64  2.3.2 Incompatibility of mutant mat A-l alleles in a transformation assay  65  2.3.3 Construction of chimerics between mat A-l and FMR1  71  2.3.4 Site-directed mutagenesis of L R R in mat A-l  74  2.3.5 Heterokaryon incompatibility function of other mating-type proteins  75  m  2.4 D I S C U S S I O N  3. Molecular Cloning and Sequencing of tol (to/erant)  77  86  3.1 I N T R O D U C T I O N  86  3.2 M A T E R I A L S A N D M E T H O D S  87  3.2.1 Strains, media and culturing methods  87  3.2.2 Strain construction  87  3.2.3 Chromosome walk and physical mapping  89  3.2.4 Transformation assay and subcloning of tol  89  3.2.5 Nucleic acid isolation and hybridization  90  3.2.6 D N A sequence analyses  90  3.3 R E S U L T S  91  3.3.1 Isolation of a cosmid spanning the tol locus  91  3.3.2 Bracketing of tol by genetic crossovers and R F L P analysis  94  3.3.3 Transformation reduction assay for T o f activity  97  3.3.4 Molecular characterization of tol sequence  97  3.3.5 Complementation of the tol mutant and D N A sequence analysis of the tol ~ allele. 101 3.4 D I S C U S S I O N  4. Characterization of tol and Isolation of top (TOL-interacting Protein) Genes  103  108  4.1 I N T R O D U C T I O N  108  4.2 M A T E R I A L S A N D M E T H O D S  110  4.2.1 Strains, media and culturing methods  110  4.2.2 tol plasmid construction and transformation assay  112  4.2.3 Isolation of foZ-RIP mutants  112  4.2.4 Nucleic acid isolation/hybridization and Reverse transcriptase P C R analysis  113  4.2.5 Yeast 2-hybrid system and screening of a c D N A / p A D - G A L 4 library  114  4.2.6 D N A sequence analyses  116  iv  4.3 R E S U L T S  116  4.3.1 Functional analysis of tol constructs  116  4.3.2 Isolation of to/-RIP mutants  118  4.3.3 Expression analysis of tol by R T - P C R  120  4.3.4 Phenotypic studies of tol- mutant as complemented by different tol constructs  122  4.3.5 Detection of tol sequences in other Neurospora species  124  4.3.6 Isolation of top (TOL-interacting Protein) genes  126  4.4 D I S C U S S I O N  5. Analysis of tof and Its relation to Non-allelic and Sexual Incompatibility  129  134  5.1 I N T R O D U C T I O N  134  5.2 M A T E R I A L S A N D M E T H O D S  138  5.2.1 Strains, media and culturing methods  138  5.2.2 Repeat-induced point (RIP) mutation  140  5.2.3 Nucleic acid isolation and hybridization  140  5.2.4 tof isolation, plasmid construction and transformation  141  5.2.5 D N A sequence analyses  142  5.3 R E S U L T S  142  5.3.1 R I P (repeat-induced point) mutation of tof  142  5.3.2 Molecular cloning and sequencing of tof  149  5.3.3 Transformation assay for tof non-allelic and mating-type incompatibility activity.151 5.3.4 Testing of sexual incompatibility function in tof 5.4 D I S C U S S I O N  152 155  6. Concluding Remarks  161  7. Bibliography  164  8. Appendices  207  8.1 Genetic crossover between his-5 and tol can be detected by cosmid G13:C8  207  8.2 physical map of cosmids containing the tol gene  208  8.3 Hydrophobicity Plot for T O L  209  8.4 Detection of vipl sequence in various Neurospora species  210  v  List of Tables Table 3-1 List of Neurospora crassa strains used in this study  88  Table 3-2 Physical ( R F L P ) mapping of the trp-4 gene (in plasmid pSV50-31:2G) and cosmids identified in the chromosome walk  93  Table 4-1 List of Neurospora crassa strains used in this study  Ill  Table 4-2 Constructs used in complementation of tol mutant  123  Table 4-3 Positive clones identified from 2-hybrid library screen using T O L as bait protein and mycelial c D N A products as target proteins  127  Table 5-1 List of Neurospora strains used in this study  139  Table 5-2 Genotypes of 50 progeny from the tol -RIP cross  144  T  vi  List of Figures Figure 1-1 Life cycle of Neurospora crassa  17  Figure 1-2 Mating type locus organization in Neurospora crassa and in Podospora anserina... 19 Figure 2-1 Strategy for transformation reduction assay for heterokaryon incompatibility  66  Figure 2-2 Results of the introduction of mutant mat A-l alleles (A ) into a and A recipient m  strains  67  Figure 2-3 Mutant and truncated mat A-l alleles: topography of presumptive translational products and their properties  68  Figure 2-4 Leucine-rich repeats in mating-type proteins  70  Figure 2-5 Mating-type amino acid comparison between Neurospora crassa M A T A - l and  Podospora anserina F M R 1  72  Figure 2-6 Results of the introduction of various constructs into a and A recipient strains  76  Figure 2-7 Miscellaneous mat A-l constructs and their functional analyses  79  Figure 3-1 Contiguous cosmids isolated from a chromosome walk around trp-4 on the right arm of linkage group I V  92  Figure 3-2 The tol locus was braketed by genetic crossovers and restriction fragment length polymorphism ( R F L P ) analysis of progeny  95, 96  Figure 3-3 Nucleotide and amino acid sequence of the tot gene of N. crassa  99  Figure 3-4 Structural features identified in T O L  100  Figure 3-5 Sequence homology among T O L and the predicted products of two het genes, het-6  of N. crassa and het-e of P. anserina  102  Figure 4-1 Functional analysis of tol deletion and frame-shift constructs  117  Figure 4-2 Phenotypic and genotypic analysis of fo/-RIP mutants  119  Figure 4-3 Expression of tol as analyzed by R T - P C R  121  Figure 4-4 Hybridization of tol to D N A from Neurospora species  125  Figure 4-5 A m i n o acid similarity between the predicted polypeptide sequence from a tip gene (7D8)and VIP1  128  Figure 5-1 R F L P analysis of tol sequences i n the progeny from the to/ -RIP cross r  Figure 5-2 A m i n o acid alignment between T O L (N. tetrasperma) and T O L (N. crassa) T  c  147 150  Figure 5-3 Mating assay for tot transformants  154  Figure 8-1 Genetic crossover between his-5 and tol as detected by G13:C8  207  Figure 8-2 Physical map for cosmids X 2 5 : D 7 and G13:18  208  Figure 8-3 Hydropathy plot of T O L as analyzed by the method of Kyte and Doolittle (1982) with a 7-residue window  209  Figure 8-4 Detection of vipl sequence in various Neurospora species  210  vn  Acknowledgements Thank G o d it's over.  viii  1. Overview and Introduction 1.1 THESIS OBJECTIVES In Neurospora crassa Shear & Dodge, mating is controlled by a single locus with two mating types, A and a. This mating-type locus has an additional unique function during vegetative growth known as heterokaryon (or vegetative) incompatibility: if hyphae of opposite mating-types fuse during the vegetative stage, the resulting heterokaryotic cells are inhibited in their growth. Fusion of hyphae with the same mating-type shows no such reaction. A n unlinked mutation tol suppresses the heterokaryon incompatibility but does not affect sexual function.  Sequencing of the two mating-type alleles, A and a, showed that the two have totally dissimilar sequences of 5301 and 3235 base pairs, respectively. A single open reading frame (ORF) from each mating-type idiomorph, mat A-l and mat a-l, confers both mating function and heterokaryon incompatibility. Both M A T A - l and M A T a-l contain conserved DNA-binding motif as shared by some other transcriptional activators and their presence/absence affect expression of many sexual developmental genes (sdv). The objectives of this thesis were to understand the roles of mat A-l and tol in mediating mating-type-associated vegetative incompatibility in N. crassa. These aims were approached by dissection of functional domains necessary for heterokaryon incompatibility of mat A-l and by the cloning and characterization of tol.  1.2 GENERAL INTRODUCTION TO NEUROSPORA CRASSA  In this section, a summary on the background of pioneering Neurospora research is given. The advantages that make Neurospora crassa (and related species) a model organism for genetic and molecular biology will also be discussed.  1  1.2.1 Neurospora crassa, a model genetic organism French microbiologists first described Neurospora more than 150 years ago. It was discovered in Paris when bread from bakeries was spoiled by massive growth of an orange mold ("le champignon rouge du pain" - the red bread fungus). A commission was set up to investigate the event and to make recommendations. The commission's report (Payen, 1843) described the morphology of the vegetative phase of the fungus. This report and later works by A. Payen (1848, 1859) were cited by Pasteur (1862) in refuting the theory of spontaneous generation.  The next round of study on Neurospora began in Java, Indonesia during the early 1900's. Attracted by a local food called oncham (a bright orange cake consisting of Neurospora grown on soybean), F.A.F.C. Went (a Dutch plant physiologist) decided to study the orange oncham fungus. His work included using Neurospora to study the effect of various substrates on different enzymes activities (Went, 1901) and the effect of light on carotenoid production (Went, 1904).  B.O. Dodge was the first to discover heat activation of ascospores (1912). This finding made Neurospora available for genetic studies. In his first Neurospora paper (Shear & Dodge, 1927), Dodge showed that hybrid perithecia from crosses between different species developed slowly and were sterile (or poorly fertile). This innovation of differentiating species based on production of fertile offspring (biological species concept), in contrast to purely morphological criteria, was later popularized by many biologists. The orange fungus was assigned to the new genus Neurospora (name based on the nerve-looking grooves on the ascospores); previously, the vegetative stage was called Oidium aurantiacum, Penicillium sitophilum and Monilia sitophila. Dodge also carried out the first tetrad analysis with Neurospora crassa (a heterothallic species), demonstrating that the eight ascospores of an ascus display a perfect Mendelian ratio (4:4) in mating-type segregation (Dodge, 1927). This finding fascinated a graduate student at a lecture  by Dodge given at Cornell University in 1930. This graduate student, G.W. Beadle, later teamed up with E.L. Tatum in working on Neurospora and obtained the first biochemical mutants.  Another Neurospora research pioneer influenced by Dodge was C C . Lindegren. As a junior associate of T.H. Morgan (founder of the genetic map unit; leader of a Drosophila group and the head of the Biology division at CalTech), Lindegren followed Dodge's advice to work on Neurospora. Lindegren (1936) worked out the first linkage maps using mating-type, centromeres, and morphological mutants. His work established the genetics of Neurospora on a sound foundation and made the orange mold an ideal choice for obtaining the first auxotrophic mutants in experiments by B.O. Beadle and E.L. Tatum.  The first advocacy of inherent defects in metabolism can be dated back to the early 1900's (Garrod, 1909). At that time, most geneticists still resisted accepting the direct relationship between genes and proteins, because they thought that development was too complex to be explained by a simple theory such as gene action (Sturtevant, 1965). It was not until Beadle and Tatum, in the early 1940's, began using Neurospora crassa as an experimental organism for biochemical genetics that the theory became testable. Neurospora was an ideal choice for obtaining auxotrophic mutants because of its rapid life cycle and its simple nutritional requirements (Butler et ah, 1941). In the Beadle-Tatum paper (1941), they described three Xray-induced mutants that grew on "complete medium", but failed to grow on "minimal medium." Their interpretation was that the mutations in these isolates affected genes required for the production of growth-essential compounds present in complete, but not minimal, medium. The three growth-essential substances were found to be pyridoxine, thiamine and p-aminobenzoic acid. Other mutants that became the focus for Beadle and Tatum's later work were arginine auxotrophs. They were of specific value because the mutations were mapped to three loci on separate chromosomes, and the three mutants had different growth responses to supplements (arginine and two related compounds, ornithine and citrulline). Beadle and Tatum proposed that 3  biochemical reactions in vivo occur as a series of discrete, stepwise reactions.  Each reaction is  specifically catalyzed by a single enzyme, which is specified by a single gene. This model, now known as the one-gene-one-enzyme hypothesis (Beadle, 1945), soon gained support from similar studies of other biosynthetic pathways and achieved general acceptance.  1.2.2 Advantages of Neurospora Neurospora is blessed with advantages for genetic and molecular research. It has over a thousand loci identified and mapped to linkage groups, 25% of which have been molecularly characterized (Perkins et al., 2000). It is a stable haploid (with a diploid zygote), and all four products of individual meioses can be recovered (Perkins & Barry, 1977). Centromeres can be mapped easily. It has low D N A content (43 megabases; 90% of the genome is of unique sequence) and the seven chromosomes can be resolved by pulse-field gel electrophoresis. In fact, the smallest chromosome of Neurospora has D N A content less than that of the Escherichia coli sequence. It has a short generation time, a fast growth rate, and large numbers of sexually derived progeny; even rare recombination events can be detected.  It has relatively simple  nutritional requirements and easy cultivation and preservation (Perkins, 1986). The chromosome cytology of N. crassa resembles that of higher plants and animals (Perkins, 1979). It also forms heterokaryons, which are very useful in studies of dominance and complementation. Tetrad analysis is also a great asset in studying meiosis, chromosomal rearrangements and recombinations. The Neurospora genome project is ongoing (with linkage groups II and V near completion) and numerous sequences and physical maps are available through the world wide web (http://www.mips.biochem.mpg.de/proj/ neurospora/; http://biology.unm.edu/~ngp/home.html;  http://fungus.genetics.uga.edu:5080).  4  1.3 FUNGAL  MATING-TYPES  Some parts from this section are published in the review by Shiu & Glass (2000).  Sexual development in fungi is initiated by the fusion of haploid individuals. However, fusion between two haploid cells only occurs if they contain non-identical mating-types. The existence of mating types was first demonstrated in 1904 when two sexually compatible cultures (+ and -) of Mucorinae (zygomycetes) were isolated (Blakeslee, 1904). It was later discovered in Ascobolus and Neurospora that mating-types are differentiated by different alleles at a single locus (Dodge, 1920; Shear & Dodge, 1927). The  mating-type locus not only governs the fusion  event between individuals but also influences directly or indirectly the expression of developmentally regulated genes. In many cases, the mating-type locus contains a gene(s) that encodes a protein with a characteristic DNA  binding domain, i.e., it resembles a transcription  factor. Many mating-type genes from different fungal species have been cloned and sequenced; a striking difference is generally found between alternative mating-type sequences. The term "idiomorph" was coined to describe these dissimilar genes occupying homologous positions, i.e. the mating-type locus. In this section, I will review recent information on mating-type loci organization and the regulation of sexual development in different fungal species, including unicellular yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, mycelial ascomycetes Neurospora crassa, Podospora anserina, Cochliobolus heterostrophus and Sordaria macrospora, heterobasidomycetes Ustilago maydis, and homobasidiomycete Coprinus cinereus and Schizophyllum commune.  5  1.3.1 Saccharomyces cerevisiae Saccharomyces cerevisiae Meyer ex Hausen is a unicellular yeast that grows vegetatively by budding of haploid cells (Herskowitz, 1988). Individual haploid cells are of either a or a mating-type. The different a and a mating-types of haploid cells are determined by genes at the MAT locus (Mortimer & Hawthorne, 1969; Herskowitz & Oshima, 1981). A cell signals its presence to another cell of the opposite mating-type by producing mating-type-specific pheromones (Fields, 1990). A n a cell produces a-factor (12-aa peptide; Betz et al, 1987) which is secreted across the cell membrane and binds to a specific a-factor receptor (STE3 protein; Hagen et al, 1986) on the surface of a cells. Similarly, a cell secretes a-factor (13-aa peptide; Stotzler et al, 1976) that binds to the a-factor-specific receptor (STE2 protein; Blumer et al, 1988) on a cells. Binding of the pheromones to their specific receptors facilitates mating by inducing a series of signal transduction events (Marsh et al, 1991) that lead to (i) the transient arrest of the cell cycle in G l (so that only a single copy of chromosomes is present; BuckingThrom et al, 1973) and (ii) the production of glycoproteins and agglutinins that enhance conjugation of opposite mating-type cells (Lipke & Kurjan, 1992; Leberer et al, 1997). The two cells fuse together into a single cell (cytogamy), followed immediately by nuclear fusion (karyogamy) to produce a diploid cell. The diploid stage is the prominent form of S. cerevisiae, which reproduces vegetatively by budding. The mating functions in the a/a diploid cells are shut off ~ diploid cells do not produce or respond to pheromone and are unable to switch mating type (Schmidt & Gutz, 1994). Upon starvation, the diploid cell stops vegetative growth and proceeds through meiosis (Klein et al, 1994). Meiosis is followed by sporulation to form an ascus containing four haploid cells, two of each mating-type (Herskowitz, 1988).  Organization and function of the MAT locus The mating-type locus MAT of S. cerevisiae is located near the centromere of chromosome III (Mortimer & Hawthorne, 1969). The mating-type of a haploid individual is 6  determined by the sequence encoded in the Y region (Astell et al, 1981). For a cells, this region contains the Ya sequences (642 bp). On the other hand, a cells contain the totally dissimilar Y a sequence (747 bp) in the MAT Y region. Flanking the Y region are the X (704 bp) and the Z l (239 bp) region. X and Z l are also found flanking two silent copies of the mating-type determining sequence (sometimes referred as the Y cassette) at the HML and the HMR locus (Klar et al, 1981). HML and HMR provide intact but unexpressed copies of mating-type genes that allow mating-type switching in yeast (see below). Although located on chromosome III, these two loci are not closely linked to MAT. Two additional homologous regions, W (732 bp) and Z2 (88 bp), are present in MAT and HML.  Two divergent transcripts, MATal and MATcc2, were identified in the MATa region (Tatchell et al, 1981). M A T a l (175-aa) is a positive transcriptional factor that activates the expression of a-specific genes, such as MFal/MFa2  (encoding a-factor precursor; Kurjan &  Herskowitz, 1982) and STE3 (encodes a-factor receptor; Sprague et al, 1983). M A T a l forms a complex with a transcriptional factor MCM1 (Passmore et al, 1988). This complex binds to upstream regulatory regions of a-specific genes and activates their expression (Ammerer, 1990). Although MCM1 can bind to the upstream promoter regions by itself, it cannot activate gene expression without M A T a l (Tan & Richmond, 1990). It has been suggested that the STE12 transcriptional factor may play a role (by interacting with MATal) in the activation of a-specific genes (Sengupta & Cochran, 1991). Although M A T a l is required for mating and a-specific phenotype, it is clearly not required for meiosis (Strathern et al, 1981).  MCM1 is capable of binding to the upstream promoter region of a-specific genes, such as MFaVMFa.2 (encoding a-factor precursor) and STE2 (a-factor receptor), and activate transcription (Tan & Richmond, 1990). Since expression of a-specific genes does not require specific activation, these genes must be switched off in a cells. This is the function of the  7  MATcc2 protein (Herskowitz, 1989). MATcc2 contains a characteristic DNA binding motif, the homeodomain (Stepherd et al, 1984). MATcc2 forms homodimer in a cells and acts in conjunction with MCM1 and two general transcriptional repressors, SSN6 and TUP1, to repress a-specific genes (Mukai et al, 1991; Keleher et al, 1992). MATal mutants display some aspecific phenotype and are able to mate (weakly) with a cells (Strathern et al, 1981). The DNA binding and protein interaction characteristics of MATcc2 has been determined both functionally and structurally (Hiscock & Kiies, 1999).  Two divergent transcripts, MATal and MATal, have also been identified in the MATa region (Tatchell et al, 1981). MATa2 has no known function in conjugation or sporulation, and is apparently dispensable (Herskowitz, 1988). MATal, together with MATa2, is important in identity in a/a diploid cells. As mentioned above, diploid cells do not produce or respond to pheromone and are unable to switch mating type. However, diploid cells can express genes required for meiosis and sporulation, such as IME-1, SP012 and SP013 (Herskowitz, 1988, 1989). All these characteristics of diploid cells are controlled by the MATa2 homodimer (which represses a-specific genes) and the al/a2 heterodimer (Goutte & Johnson, 1988). MATalMATa2 heterodimer (with SSN6/TUP1) represses the transcription of MATal (hence repressing a-specific genes) and other haploid-specific genes (Dranginis, 1990), such as HO (involved in mating-type switching; Schmidt & Gutz, 1994) and RME1 (repressor of meiosis; Covitz et al, 1991).  Mating-type silencing and switching in Saccharomyces cerevisiae As mentioned above, HML and HMR regions also contain mating-type cassette Y (most strains are HMLa HMRa), in addition to that found in the MAT locus. However, the mating-type cassettes in HML and HMR are silent. Repression of the silent mating-type cassettes in HML and HMR is mediated by many genes. Among these are frans-acting SIR1 to SIR4 proteins, each of 8  which is required for repression of HML and HMR (Ivy et al, 1986; Rine & Herskowitz, 1987; Chien etal, 1993; Triolo & Sternglanz, 1996; Hecht etal, 1996; Strahl-Bolsinger, 1997). Two ds-acting sites required for H M silencing, E and I (called the silencers), flank HML and HMR but not MAT (Feldman et al, 1984; Mahoney & Broach, 1989; Loo & Rine, 1994). These e x acting elements, together with histone proteins, SIR1-4, RAP1 and ORC (origin recognition complex), form a short region of highly ordered heterochromatin-like structure resulting in the repression of HML and HMR (Loo & Rine, 1995; Weiss & Simpson, 1998; Haber, 1998). It has been suggested that D N A replication may play a role in maintenance of silencing, which is supported by the following evidence: (i) E and I contain binding sites for the transcriptional factors RAP1 and ABF1, (ii) E and I exhibit ARS activity and (iii) repression at both HML and HMR requires passage through S phase of the cell cycle (Miller & Nasmyth, 1984). However, recent experiments have provided evidence that the S phase requirement may be due to enzymes specific for S phase rather than replication (Bi & Broach, 1997; Fox et al, 1997).  Mating-type switching in yeast has been observed for decades (Oeser, 1962; Strathern & Herskowitz, 1979). In the process of mating-type interconversion, a copy of the mating-type cassette (Ya or Ya) is transposed from HMR or HML into MAT. The switch is initiated by a double-strand break (DBS) in the Z l region, 3 bp to the right of Y (Raveh et al, 1989). The DBS is introduced by an endonuclease (encoded by HO; defects in HO lead to heterothallism) during G l phase of the cell cycle (Kostriken et al, 1983; Herskowitz, 1989). A symmetric priming model has been proposed to explain the switching mechanism (which is related to gene conversion) and how the D N A originally present in MAT is degraded (Klar et al, 1984a). The phase specificity of DBS can be explained by the fact that the transcription of HO is restricted to the G l phase by several regulatory genes, including 6 SWI genes and 3 SIN genes (Herskowitz et al, 1992). Other genes required for completion of switching include those involved in D N A damage repair and recombination, such as the RAD genes (Game, 1983; Weeda et al, 1993; Schmidt & Gutz, 1994). DSBs at MAT are lethal if they are not healed by a switching event, i.e.,  9  S. cerevisiae cannot repair //Oinduced DSBs without mating-type switching (Klar et al, 1984a, b).  In S. cerevisiae, only experienced cells, i.e., those cells which have budded off at least one daughter cell, can produce switched progeny (Strathern & Herskowitz, 1979). Both progeny of a cell (the mother and the daughter) are switched whenever a switch occurs (Klar, 1989). Switching in 5. cerevisiae is very efficient, i.e., a cassette is preferentially replaced by a cassette with opposite mating-type. A recombination enhancer (RE) is the molecular basis for this directionality (Wu & Haber, 1996). In MATa cells, the RE is activated, thus allowing the left arm of chromosome III (HMLa) to be the preferred donor. In MATa cells, RE activity is repressed (by the binding of MATa2-MCMl; Wu et al, 1998a) and the entire left arm (and part of right arm) is "cold" and thus HMRa becomes the preferred donor.  Pheromone response and signal transduction The a-factor of 5. cerevisiae is encoded by two unlinked genes, MFal and MFa2 (Kurjan & Herskowitz, 1982). Each gene contains 6 tandem copies of the a-factor precursor sequence: WHWLQ(N)LK(R)PGQMY (5 perfect repeats and 1 repeat with conservative substitutions as shown in parentheses; Singh et al, 1983). The prepropheromone is transported to the endoplasmic reticulum and is subject to multiple rounds of proteolytic actions. The mature a-factor is exported by the typical secretory pathway via the golgi apparatus (Julius et al, 1984). Similarly, the a-factor precursor is encoded by two unlinked structural genes, MFal and MFa2 (Brake et al, 1985). Both precursor polypeptides contain a single copy of a-factor near the carboxy-terminus (Betz et al, 1987), which terminates in a CAAX motif (C = cysteine, A = aliphatic amino acid, and X = any amino acid). CAAX motifs have been identified as a consensus signal for isoprenylation of cysteine (Powers et al, 1986). Maturation of a-factor requires multiple post-translational modification steps. Unlike a-factor, the export of a-factor does not depend on the typical secretory pathway (Sterne & Thorner, 1987). The secretion of a10  factor requires the STE6 gene, which encodes a membrane-bound, ATP-dependent transport protein (Kuchler et al, 1989). These observations suggest that the a-factor is exported by a novel secretory pathway.  As mentioned above, binding of the pheromones to their specific receptors facilitates mating by inducing a series of signal transduction events that lead to the transient arrest of the cell cycle in G l and the induction of conjugation-specific genes. The pheromone signal transduction pathway is initiated by binding of pheromone to its receptor in the cytoplasmic membrane (Marsh et al, 1991; Duntze et al., 1994). The signal transduction machinery appears to be the same for both a- and a-factor after this step. The pheromone-binding signal, via coupling of a heterotrimeric G-protein, in which the P (STE4) and y (STE18) subunits are believed to be the active signal transmitter, activates a cascade of MAP kinase phosphorylation in this order: STE11 -> STE7 -> KSS1/FUS3 (Peter et al, 1993; Cook et al., 1997). The FUS3 and KSS1 (in the absence of FUS3) kinase phosphorylates the transcriptional factor STE12 (Madhani et al., 1997), which in phosphorylated form binds and activates the transcription of many conjugation-specific genes, including AGA1 and AGal (cell agglutination), KAR3 (nuclear fusion), STE2 and STE3 (receptors for a- and a-factor), SCG1/STE4/STE18 (structural genes of G-protein), and MFal and MFal (structural genes of the pheromone precursors) (Marsh et al, 1991; Kurjan, 1993). The activation of STE12 is more complicated than once thought and may involve its dissociation from two inhibitors (RST1 and RST2; Cook et al, 1996; Tedford et al, 1997). On the other hand, the FUS3 kinase phosphorylates FAR1, thus enabling it to inhibit the CDC28-CLN2 kinase complex, probably by causing degradation of CLN2 (Peter et al, 1993). The inhibition of the CDC28-CLN2, which controls cell cycle progression in 5. cerevisiae, is believed to cause arrest of cells in G l (Futcher, 1991). The mechanism for activation of this MAPK cascade is unknown, however, it may involve action of STE5 scaffold protein and STE20 serine/threonine protein kinase (Banuett, 1998).  11  If a cell cannot find a partner after pheromone induction, it can recover from pheromone action and resume growth by the following mechanisms: (i) internalization/degradation of occupied receptor (Chvatchko et al, 1986; Jeness & Spatrick, 1986), (ii) phosphorylation of the C-terminus of the receptor protein, which interferes with the ability of the receptor to transmit the signal (Konopka et al, 1988; Reneke et al, 1988), (iii) degradation of a- and a-factor (MacKay et al, 1988; Marcus et al, 1991), (iv) de-sensitization of the cell to pheromone by the RGS (regulator of G-protein signalling) protein, SST2, through regulation of G a GTPase activity (Dohlman et al, 1996; Apanovitch et al, 1998).  1.3.2 Schizosaccharomyces pombe Schizosaccharomyces pombe Lindner is a unicellular yeast that grows by tip extension and divides by medial fission (Egel, 1989). There are two mating-types in S. pombe haploid cells: P (plus) and M (minus) (Leupold, 1958). Under starvation, cells of opposite mating-type elongate to each other through the actions of pheromones (Leupold, 1987). The cells arrest in G l and agglutinate. Unlike S. cerevisiae, meiosis occurs immediately after cell fusion and karyogamy, results in the formation of a four-spored ascus. Two of the spores are of each mating-type.  Mating-type locus and sexual cycle in S. pombe The mating-type locus organization in 5. pombe is similar to that of 5. cerevisiae (Kelly et al, 1988). The two different mating-types are determined by genes at a single locus on the right arm of chromosome II, mat-1 (matl-P for P cells and matl-M for M cells). The P and M mating-type alleles contain dissimilar DNA sequences of 1104 and 1128 bp respectively. Two additional loci that contain silent copies of mating-type sequence, mat-2 and mat-3, are also present on chromosome II. The three loci are linked, with a 15-kb sequence separating each neighboring locus. Surrounding the mat-1, mat-2 and mat-3 loci are short regions of homology, HI (59 bp) and H2 (135 bp). An additional sequence, H3 (57 bp), is present at mat-2 and mat-3. 12  Two transcripts, matl-Pc and matl-Mc (c = conjugation), one from each mating-type, are detectable during vegetative stage (Kelly et al, 1988). Mutations in these genes lead to defects in conjugation. The predicted polypeptides of MATl-Pc (118 aa) and MATl-Mc (181 aa) are basic, a characteristic of DNA binding protein. MATl-Pc encodes a cd-domain protein (Coppin et al, 1997). MATl-Mc protein contains a DNA binding domain known as HMG box (Ner, 1992), which is found in the SRY protein that determines maleness in humans and mice (Sinclair et al, 1990). The function of MATl-Pc and MATl-Mc is proposed to be analogous to that of M A T a l , a transcriptional factor in S. cerevisiae that controls the expression of conjugationspecific genes, including those encoding pheromones and pheromone receptors. MATl-Mc activates Af-specific genes by recruiting the ubiquitous HMG transcription factor STE11 to binding sites (Kjaerulff et al, 1997). MATl-Pc, in association with a MCMl-like transcriptional factor (MAPI), controls expression of f-specific genes (Nielson et al, 1996). Unlike M A T a l , MATl-Pc and MATl-Mc are required for conjugation as well as sporulation (Kelly et al, 1988; Wilier et al, 1995; Hiscock & Kiies, 1999). Expression of matl-Pc and matl-Mc is induced by nitrogen limitation (which in turn lowers the level of intracellular cAMP; Mochizuki & Yamamoto, 1992).  Two additional mating-type transcripts, matl-Pm (from matl-P; m = meiosis) and matlMm (from matl-M) are detectable under starvation. Both transcripts are required for meiosis and sporulation, but not conjugation. MATl-Mm (42 aa) bears no striking amino acid resemblance with other mating-type proteins, although the mat 1-Mm gene contains DNA sequence similarity (at its 3' end) with that of the matl-Pc gene. The homologous region does not affect amino acid similarity between the two proteins because they are out of frame (Kelly et al, 1988). MAT1Pm contains homology with the homeodomain of MATal and MATa2, suggesting that it may have similar DNA-binding properties. MAT-Pm is a transcriptional factor required for the expression of a meiosis-specific genes, mei3 (McLeod et al, 1987; Van Heeckeren et al, 1998). 13  MED binds to and inhibits PAT1, a protein kinase that serves as a negative regulator of meiosis (also known as RANI) (see below; McLeod & Beach, 1988).  The sexual cycle in S. pombe is thought to be regulated by a 3-step progressive inactivation of the PAT1 (Nielson & Egel, 1990). Nitrogen starvation triggers the first step of inactivation and results in the expression of STE11, a HMG-type transcriptional factor required to express the matl-Pc and matl-Mc genes (Sugimoto et al, 1991). In P cells, matl-Pc and the uncharacterized mapl genes are required for the pheromone (P-factor) response pathway. The binding of M-factor (pheromone produced by M cells) to P cells triggers the second stage of the PAT1 inactivation (as mediated by STE6-induced RAS protein; Hughes et al, 1990), and results in the expression of matl-Pm and other conjugation-specific genes, e.g.,fusl (Nielson & Egel, 1990) . The regulatory pathway in M cells is believed to be similar to that of P cells. However, expression of matl-Mm is independent of matl-Mc and is not pheromone induced. There is evidence that matl-Mc regulates the pheromone receptor gene, maml (Kitamura & Shimoda, 1991) .  The M-factor pheromone is a modified nanopeptide: YTPKVPYMC (with Sfarnesylation and carboxy-methylation at the cysteine rsidue) (Davey, 1991, 1992). The Cterminus contains a CAAX box, which is also found in a-factor of 5. cerevisiae. M-factor is secreted through an ABC transporter (MAM1; Christensen et al, 1997). The P-factor is a 23-aa unmodified polypeptide, which is a KEX2-cleavage product of its four-repeat precursor (Davey et al, 1994; Imai & Yamamoto, 1994). The M-factor is encoded by 3 redundant genes, MFml to MFm3 (Davey, 1992; Kjaerulff et al, 1994) while P-factor is encoded by mapl (Imai & Yamamoto, 1994). The M-factor receptor and the P-factor receptor are encoded by maml (Kitamura & Shimoda, 1991) and map3, respectively (Tanaka et al, 1993). Both receptors contain seven transmembrane domains, which characterize them as the rhodopsin/adrenergic type receptors (Dohlmann et al, 1991). MAM2 is homologous to a-factor receptor of 5. 14  cerevisiae (STE2) and pheromone receptors of U. maydis (PRA1 and PRA2). All these receptors recognize farnesylated mating factors (Schafer & Rine, 1992). MAP3 is homologous to a-factor receptor of S. cerevisiae (STE3). The pheromone binding signal in S. pombe is transmitted by G-protein, with the Got subunit playing the active role in the MAP kinase signal transduction (Obara et al, 1991; Banuett, 1998). Unlike S. cerevisiae, the mating response pathway in 5. pombe is dependent upon ras and nitrogen starvation (Hiscock & Kiies, 1999).  Mating-type silencing and switching in S. pombe In 5. pombe, the mating-type cassettes in mat-2 and mat-3 are silent. Mutants were found in which the silent mat-2 and mat-3 cassettes are expressed. These include clrl to clr4, swi6 and rikl (Thon & Klar, 1992; Lorentz et al, 1992; Ekwall & Ruusala, 1994). Meiotic recombination in the K region between mat-2 and mat-3 (which normally does not occur; Grewal & Klar, 1997) is permitted in some of these mutants (Egel et al, 1989). The silencing of mat-2 and mat-3 is believed to be mediated by heterochromatin formation in these loci (Lorentz et al, 1994; Allshire et al, 1995; Ekwall et al, 1996). SWI6 contains a chromatin organization modifier domain and might represent the structural component of the chromatin. Cw-acting sequences flanking mat-2 are required for effective blockage of transcription (Ekwall et al, 1991).  Switching of mating-type in 5. pombe involves the transfer of a mating-type cassette from mat-2 or mat-3 to the mat-1 locus. The transfer is initiated by a double-strand break (DSB) in the HI homology box of mat-1 at the border to the mating-type-specific sequence (Nielsen & Egel, 1989). Binding of SAP1 protein at SAS1 (see below) is necessary for generation of DSB (Arcangioli & Klar, 1991). DSBs are detectable during all stages of the cell cycle (c.f. DSBs detectable only in G l in S. cerevisiae). In S. pombe, only daughter cells show a converted mating-type (Egel, 1984; Klar 1992a, b; c.f. both progeny show switching in S. cerevisiae). This imprinting is due to strand-specific DNA modification at the matl locus (Dalgaard & Klar, 1999). 15  There are three classes of switching-defective mutations that define fran-active genes required for recombination (Egel et al, 1984; Klar, 1992b): (i) Class la (swil, swi3, swiT) represent the genes required for making a DSB at mat-1, (ii) Class lb (swi2, swi5, swi6) represent the genes involved in the utilization of the DSB for recombination, and (iii) Class II (swi4, swi8, swi9, swilO, rad 22) represent the genes required for general recombination and repair. In addition, at least two cis-acting sites, SAS1 and SAS2 (which span 150 bp on the left of HI at the mat-1 locus), are required for the generation of DSB (Klar et al, 1991). Switching directionality {i.e. donor is preferentially selected from the locus containing the opposite matingtype cassette) is observed in 5. pombe. One explanation for this effect is that the chromatin structure of the donor loci is altered between P and M cells, therefore promoting preferential selection of the opposite mating-type cassette (Klar, 1992b).  1.3.3 Neurospora crassa The life cycle of Neurospora crassa Shear & Dodge begins when a mature ascospore, upon heat activation, germinates and produces a multinucleate mycelium (Fincham et al, 1979; Figure 1-1). Under nitrogen starvation, the female reproductive structures (protoperithecia) are formed (Johnson, 1978). The outer layer of the protoperithecium is a wall of hyphae. Inside is a coiled hypha, the ascogonium, from which projects female receptive hyphae called trichogynes. Mating occurs between these trichogynes and a target cell (conidium, microconidium, or hypha) of the opposite mating-type (either A or a) which is destined to function as the male. Pheromone emitted by the male cell directs the growth of trichogynes (of opposite mating-type) toward them (Bistis, 1981). Once fertilized, the protoperithecium develops into the perithecium.  16  Figure 1-1. Life cycle of Neurospora crassa. Reprinted from Fincham et al. (1979).  The male nuclei, presumably under their own genetic control (Vigfusson et al., 1971), travel down the trichogyne, where one nucleus enters the ascogonium and becomes associated with the female nucleus. A series of synchronous nuclear divisions, accompanied by the darkening and the enlargement of the perithecium, gives rise to a cluster of dikaryotic ascogenous hyphae. After 8-10 divisions, karyogamy occurs in the penultimate cells of the ascogenous hyphae, followed by two meiotic divisions and a postmeiotic mitosis. The final products are perithecia containing many mature asci, each housing eight ellipsoidal ascospores that are shot through an opening in the perithecial beak (Fincham et ah, 1979).  Mating-type locus organization in Neurospora crassa In N. crassa, mating is controlled by a single locus (mat) with two mating-types, A and a (Figure 1-2; Shear & Dodge, 1927; Perkins et al., 1976).  The mat locus, located on the left arm  of linkage group I, has an additional unique function in vegetative growth known as heterokaryon incompatibility. Heterokaryon incompatibility, a process that eliminates heterokaryon formation between fungi with genetically distinct nuclei, is controlled by mat and at least 10 other het loci in N. crassa (Saupe, 2000; Glass et al., 2000; see section 1.4). An allelic difference at the mat locus leads to death and compartmentation of cells along the line of fusion between hyphae of opposite mating-types (Beadle & Coonradt, 1944). Early attempts to separate the heterokaryon incompatibility function and the mating function of A and a idiomorphs by genetic recombination were unsuccessful (Newmeyer et al, 1973), indicating that either the two functions are conferred by the same gene, or they are encoded by separate genes that are closely linked. An unlinked locus tol was discovered that suppressed the vegetative incompatibility without affecting mating function (Newmeyer, 1970).  A cosmid containing the N. crassa A mating-type locus {mat A) was first isolated by complementation of un-3 (closely linked to mat) activity (Glass et al., 1988). The 5.3 kb mat A  18  I. Neurospora crassa A Idlomorph 5301 bp  mt A-3 mt A-2 poat-fartlllzatlon lunotlons  mt A-1 hetarokaryon Incompatibility futility  a Idlomorph 3236 bp  mt a-1 hatarokaryon Incompatibility lartlllty  II. Podospora anserina mat- Idlomorph 4700 bp -POOOOQO<xSgg<3  txxxxxx>oo55gl8MR2 BMR1 poat-tertlllzatlon funotlona  FMR1 lartlllty  mat+ idlomorph 3800 bp  FPH1 fertility  Figure 1-2. Mating type locus organization in Neurospora crassa and in Podospora  anserina.  Reprinted from Glass & Nelson (1994).  19  locus, which contains three ORFs: mat A-l, A-2 and A-3. The mat A-l gene is both necessary and sufficient to confer mating specificity of A and trigger heterokaryon incompatibility with a (see below for heterokaryon incompatibility) (Glass et al, 1990). MAT A - l protein is believed to be a transcriptional factor (although direct transcriptional activity has not been shown). It contains an a l (sometimes called a-) domain that is shared by other ascomycetes mating-type proteins, namely M A T a l (S. cerevisiae) (Astell et al., 1981), MATl-Pc (5. pombe) (Kelly et al, 1988; Coppin et al, 1997), FMR-1 (P. anserina) (Debuchy & Coppin, 1992), and MAT-1 (C. heterostrophus) (Turgeon et al, 1993). The a l domain of M A T a l protein is proposed to interact with MCM1 in S. cerevisiae to recognize promoters of a-specific genes (Grayhack, 1992; Yuan et al, 1993). The first 85 amino acids of MAT A - l (includes the a l domain) is sufficient for low female fertility but the entire polypeptide is required for male and full female fertility (Saupe et al, 1996b).  MAT A - l also plays a role in the post-fertilization functions. When a functional mat A-l copy is transformed to an ectopic site in a mat A-l mutant, the transformants gain mating activity as A but only produce barren perithecia after a cross (Glass et al, 1990; Saupe et al, 1996b; Vellani, 1988). This observation shows two important phenomena: (i) MAT A - l is required for sexual development after mating. MAT A - l protein could be required for nuclear identity for conjugate mitotic divisions of opposite mating-type nuclei or nuclear transport into the ascogonium. (ii) The mat A-l gene has to be located at the mat locus for completion of the sexual cycle. The mechanism for physical location requirement for mat A-l is unknown (note: the ectopic restriction also applies to mat a-l). It is possible that the mat locus gives a strict expression pattern for mat A-l to function during sexual development. Alternatively, matingtype genes may be required to pair during the sexual cycle. Transvection (regulation dependent on pairing of alleles; Wu & Morris, 1999) between alleles of the Asm-1 locus has been reported to affect meiosis (Aramayo & Metzenberg, 1996).  20  Two more ORFs were identified within the A idiomorph (Glass & Lee, 1992). Mutations in matA-2 and matA-3 do not affect mating or heterokaryon incompatibility, but affect ascosporogenesis; a matA-2 and matA-3 double mutant makes few asci with ascospores (the development of the perithecia is arrested 3-4 days after fertilization). Hence, the two ORFs may govern ascogenous hyphal development. Interestingly, the mat A-2 and mat A-3 single mutants give abundant biparental asci when crossed with a, indicating that MAT A-2 and MAT A-3 could be redundant and may function in the same pathway (Ferreira et ah, 1998). Since matA21mat A-3 double mutants give rare asci that are biparental (instead of uniparental "selfish" asci; see section 1.3.4), it has been suggested MAT A-2 and A-3 are involved in the synchronous dikaryon proliferation in the ascogenous hyphae, and not in the migration of opposite matingtype nuclei to the crozier (Ferreira et al., 1998).  MAT A-2 is not similar to any known protein except the P. anserina SMR1 mating-type protein (see section 1.3.4) (Ferreira et al., 1996). MAT A-2 and SMR1 contain a highly conserved amphipathic a-helical motif that may specify a new DNA binding motif (Debuchy et al., 1993). MAT A-3 contains a HMG (high mobility group) domain (Bianchi & Beltrame, 1998). HMG domain proteins bind to the minor groove and introduce bends in DNA (Bewley et al., 1998). MAT A-3 binds to the same DNA fragments in vitro as MAT a-1 does (see below) (Kronstad & Staben, 1997). Although there is no evidence for direct regulation by these matingtype proteins of downstream genes, it has been shown that expression of mating pheromones (D. Ebbole, personal communication) and at least two sexual developmental (sdv) genes (Nelson & Metzenberg, 1992; Nelson et al., 1997; Ferreira et al., 1998) are under the control of A , crassa 7  mating-type polypeptides. The size and expression pattern of transcripts adjacent to the MAT locus is also found to be mating type-specific (Randall & Metzenberg, 1995,1998).  The a idiomorph contains two ORFs - mat a-1 (Staben & Yanofsky, 1990) and mat a-2 (Pbggeler & Kiick, 2000). Griffiths and DeLange (1978) obtained 25 mat a mutants. All but one 21  of the mutants had lost both mating and heterokaryon incompatibility functions. The exceptional m33  a  m u t a n t  j  o s t  on  jy  m  e  heterokaryon incompatibility function. MAT a-l is necessary and  sufficient for all mating type functions for a (mating, post-fertilization, heterokaryon incompatibility), since replacement of the A idiomorph with a construct containing only mat a-l can switch mating-type (Chang & Staben, 1994). mat a-2 was identified by transciptional analysis and may represent a non-essential gene or a non-translated pseudogene (Poggeler & Kiick, 2000). MAT a-l has an HMG box and binds to DNA in vitro centering on the sequence CAAAG in common with binding sites of other HMG proteins (Philley & Staben, 1994; Kronstad & Staben, 1997). Mating function of MAT a-l correlates with DNA binding activity; deletion within the HMG box eliminated both DNA binding in vitro and mating in vivo, but did not affect heterokaryon incompatibility (Philley & Staben, 1994). Unlike MAT A - l , MAT a-l is apparently required after karyogamy, since RIP (Selker, 1990) mutants in mat a-l cannot be isolated (Kronstad & Staben, 1997). MAT a-l is also believed to play a role in dikaryon proliferation since strains bearing duplication in mat a-l always resolve after a cross (Chang & Staben, 1994). Preliminary yeast two-hybrid data showed that MAT a-l interacts with MAT A - l (Badgett & Staben, 1999). In yeast, the MATal-a2 heterodimerization of the mating type proteins is important in the repression of haploid mating-specific genes and de-repression of meiotic genes (Johnson, 1995; Johnson et al., 1998). However, heterodimerization of MAT A-l and MAT a-l may be relevant to heterokaryon incompatibility rather than to sexual development; a mutant MAT a-l protein lacking interaction activity with MAT A - l affected heterokaryon incompatibility but not sexual function (C. Staben, personal communication). In P. anserina, mating-type-associated heterokaryon incompatibility does not exist; a yeast two-hybrid experiment failed to demonstrate interaction between FMR1 and FPR1 (homologs of MAT A - l and MAT a-l, respectively) (Coppin et al., 1997). These data suggested that an al/oc2-type heteromeric complex may not be required for meiosis function in filamentous ascomycetes.  22  Although the pheromone response pathway of N. crassa has not been characterized, proteins similar to components of the yeast MAP kinase pheromone pathway have been identified, such as mating pheromones (MFA and MFa; D. Ebbole & D. Bell-Pedersen, unpublished data), a heterotrimeric G proteins (Turner & Borkovich, 1993, unpublished data), a STE11 homolog (NRC-1; Kothe & Free, 1998), a FUS3 homolog (MAK-2; P. Bobrowicz & D. Ebbole, unpublished data) and a STE12 homolog (Neurospora Genome Project; http://biology.unm.edu/~ngp/home.html).  1.3.4 Podospora anserina Podospora anserina (cas.) Niessl is in the same family (Sordariaecae) as N. crassa, although it displays a different sexual life style (Esser, 1974). P. anserina is a pseudohomothallic ascomycete, with opposite mating-type nuclei (mat+ and mat-) compartmentalized within a single ascospore, yielding functional self-fertility. The mating-type locus organization of P. anserina is very similar to that of N. crassa. The mat- and mat+ idiomorphs are composed of 4.7 kb and 3.8 kb of dissimilar DNA sequence, respectively (Debuchy & Coppin, 1992). Deletion of the mat locus (matO) results in sterility, but has no effect on vegetative growth or formation of female reproductive organs (Coppin et al., 1993). Transformation of the entire mat locus into ectopic locations in a matO strain results in full complementation of mating and ascospore formation (Coppin et al., 1993). Hence the positional effect observed in N. crassa (ectopic mating-type genes cannot complement mat mutants in postfertilization functions) does not apply to P. anserina.  Mating-type genes of P. anserina (Debuchy & Coppin, 1992; Debuchy et al., 1993) are homologous to those of N. crassa (Glass et al., 1990; Ferreira et al., 1996) (Figure 1-2). The mat- mating type was identified by heterologous hybridization using N. crassa mat A sequences (Picard et al, 1991). FPR1 is a homolog of MAT a-1, while FMR1, SMR1 and SMR2 are homologs of MAT A - l , MAT A-2 and MAT A-3, respectively. Mating-type genes from P. 23  anserina and N. crassa are interchangeable for mating function; FMR1 confers A mating activity in N. crassa while mat A-l confers mat- mating activity in P. anserina (Arnaise et al., 1993). However, functional conservation for heterokaryon incompatibility and post-fertilization events are not observed; when FMR1 and FPR1 were introduced into a N. crassa background, an incompatibility reaction was not induced (Arnaise et al., 1993). FMR1 (mat-) and FPR1 (mat+) are involved in mating function much like MAT A - l and MAT a-1, while SMR1 and SMR2 are involved in fruiting bodies maturation and ascospore formation (Debuchy et al., 1993).  Internuclear complementation shows that SMR1 is not a bona fide mating type protein, since SMR1 expression is not nucleus limited and that it can function in a mat-, mat+ or both nuclei (Arnaise et al., 1997). Since mutations in SMR1 lead to barren perithecia with no ascogenous hyphae, SMR1 is likely to be responsible for initial development of biparental ascogenous hyphae, after the nuclear recognition step (Arnaise et al., 1997). There is evidence SMR1 does so by lifting the growth arrest of biparental ascogenous hyphae after mat+ and marhave paired and thus allow proliferation. This hypothesis was based on the observation that mat+ ascospores carrying constitutively transcribed FMR1 and SMR2 are unable to germinate, but ascospore lethality can be suppressed by the over-expression of SMR1 (Coppin & Debuchy, 2000).  The bona fide mating type proteins in mat-, FMR1 and SMR2, physically interact with each other in a yeast two-hybrid system (cited in Coppin et al., 1997). The two proteins are possibly involved in nuclear recognition since they are required to ensure biparental dikaryotic state after fertilization (Zickler et ah, 1995). Loss-of-function mutations in FMR1 or SMR2 lead to the formation of monokaryotic progeny and uniparental dikaryotic progeny (Zickler et al., 1995). Since FMR1 is not likely to be nuclear limited while SMR2 is, it was proposed that SMR2 acts as a carrier protein that directs FMR1 to the mat- nucleus (Arnaise et al., 1997). FPR1 is apparently limited to the mat+ nucleus (Arnaise et al., 1997). Debuchy (1999) has 24  proposed a model that explains the roles of mating-type proteins in nuclear recognition during formation of dikaryotic ascogenous hyphae. Internuclear recognition and nuclear positioning is proposed to be controlled by nucleus-limited expression of mating-type gene products: each nucleus controls the expression and localization of mating-type-specific pheromone/receptors in its cell wall and plasmalemma (Debuchy, 1999). Spatial cues are generated when the two opposite mat nuclei are close enough to superimpose their specific expression domains and therefore allowing the binding of pheromones to their respective receptors. This cue in turn triggers the migration of the opposite mat nuclei into the ascogenous hyphae, probably by reorganization of the cytoskeleton and secretory apparatus as described in yeast (Drubin & Nelson, 1996; Thompson-Coffe & Zickler, 1994; Berteaux-Lecellier et al, 1998; Inoue et al, 1998). The uniparental progeny observed in a SMR2 mutant can be explained with the model that the SMR2/FMR1 nuclear recognition system is no longer functional, and that it allows contaminating mat+ pheromones to trigger a spatial cue for mat- nuclei to migrate to the ascogenous hyphae (Debuchy, 1999).  1.3.5 Cochliobolus heterostrophus The C. heterostrophus (Drechsler) Drechsler mating-type idiomorphs are much smaller than that of N. crassa and P. anserina. Each idiomorph only contains one mating-type gene, MAT-1 (1297 bp) or MAT-2 (1171 bp) (Turgeon etal, 1993). The MAT-1 gene was isolated based on the dual-mating phenotype it produces when transformed into a MAT-2 strain. MAT-1 contains the a l domain that is found in some other fungal mating-type proteins (Coppin et al, 1997). MAT-2 contains an HMG DNA binding domain that is shared by other fungal mating type proteins, MAT a-l (Staben & Yanofsky, 1990), MAT A-3 (Ferreira et al, 1996), FPR1 (Debuchy & Coppin, 1992), SMR2 (Debuchy et al, 1993) and MATl-Mc (Kelly et al, 1988). Interestingly, the position of the intron is conserved in the above-mentioned a l and HMG families (Coppin et al, 1997). MAT-1 and MAT-2 have a small island of nucleotide sequence similarity, suggesting they may share a common origin (Coppin et al, 1997; Turgeon, 1998). 25  Although the mating type sequences of C. heterostrophus (a loculoascomycete) do not share extensive similarity with those of pyrenomycetes (N. crassa and P. anserina), introduction of either MAT-1 or MAT-2 into a P. anserina mating-type deletion strain confers mating activity (Coppin et al., 1997), showing that the function of mating-type genes are conserved between these diverse fungi. Homologs of MAT A-27 SMR1 and MAT A-3/SMR2 were not found in C. heterostrophus. Genes flanking the MAT locus are not involved in the mating process and MAT1 and MAT-2 alone confer full mating and post-fertilization functions (Wirsel et al., 1998).  Transcript analysis reveals special features of C. heterostrophus mating type system. Unlike the other filamentous ascomycetes, the MAT-l/MAT-2  idiomorphs encode transcripts  which start 5' and end 3' within sequences common to both mating types (Leubner-Metzger et al., 1997). MAT transcripts are controlled and can be detected in cultures grown in minimal medium but not in complete medium (Leubner-Metzger et al, 1997). Transcriptional and translational controls may also play a role in MAT gene expression during sexual development (i) optional splicing of the two 5' untranslated introns and at least two transcription start sites result in three types of transcripts (although deletion of the 5' end does not interfere with crossing ability) (Wirsel et al, 1998; Leubner-Metzger et al, 1997); (ii) 160 bp of 3' UTR is required for ascospore production (Wirsel et al, 1998). The 3' MAT UTR region can potentially provide localized expression of MAT genes so that the two opposite mating type nuclei can distinguish one and another (Wirsel et al, 1998). Unlike N. crassa (Chang & Staben, 1994), expression of MAT genes specifically at the MAT locus is not required for ascospore formation in C. heterostrophus (Wirsel et al, 1996). However, interference by ectopic MAT transgenes with the resident MAT gene function (at the MAT locus) is observed in N. crassa, P. anserina and C. heterostrophus (Chang & Staben, 1994; Coppin et al, 1993; Wirsel et al, 1996).  26  MAT-2-like genes have been isolated from several Cochliobolus spp. (and related loculoascomycete genera) using primers matching C. heterostrophus sequences (Arie et al., 1996; Turgeon et al., 1995; Sharon et al, 1996). Despite the possession of a MAT-2 homolog (which is functional as a transgene in C. heterostrophus), Bipolaris sacchari (sugarcane pathogen related to Cochliobolus spp.) remains an asexual fungus (Sharon et al., 1996). The transformation of C. heterostrophus MAT genes to B. sacchari does not introduce selfing or crossing ability with other B. sacchari strains, but transformants can initiate (but not complete) sexual development with C. heterostrophus. The asexual nature of B. sacchari therefore lies in the mutation of some non-mating-type gene(s), and there is a potential for an asexual fungus to gain sexual development. A mating type-like locus with functional mat genes has also been reported in another asexual fungus, Candida albicans (Hull & Johnson, 1999).  Although transformation of an asexual fungus to a sexual fungus has not been achieved, the conversion of a heterothallic C. heterostrophus MAT-deletion strain into a self-fertile homothallic strain has been achieved by the expression of a natural MAT-l/MAT-2 fusion gene from the homothallic C. luttrellii (Yun et ah, 1999). This conversion plus the analysis of the recombination points in the fusion sequence provides evidence that a homothallic species may arise from an uneven crossover in a heterothallic species. This gives support to the speculation that heterothallic species are ancestral to homothallic species and that a single recombination event between the two mating types can lead to this transition (Yun et al., 1999).  1.3.6 Sordaria macrospora S. macrospora (a pyrenomycete) is closely related to N. crassa and P. anserina. However, it is a true homothallic species - a single strain of S. macrospora containing one type of nucleus can enter the sexual pathway without a partner. The mating type locus of S. macrospora contains 4 ORFs, Smta-1, SmtA-1, SmtA-2 and SmtA-3, which were named according to their homology with the N. crassa mating type genes (Pbggeler et al, 1997). These 27  mating type genes are present in a single-copy and are transcriptionally expressed. In contrast, the presence of all four mating type genes is not demonstrated in other homothallic Sordariaceae (Glass et al, 1988; Cisar et al, 1994; Glass et al, 1994; Beatty et al, 1994; Glass & Smith, 1994). S. macrospora mating type proteins have strong sequence similarity to the counterparts found in N. crassa (Glass et al, 1990; Ferreira et al, 1996) and P. anserina (Debuchy & Coppin, 1992; Debuchy et al, 1993) with the exception of smta-1 and smtA-3. Although a conserved HMG box is found, SMTa-1 lacks 100 amino acids at the C-terminus. The Smta-3 gene contains sequence similarity to both N. crassa A and a idiomorphs. In SMTA-3, the HMG box is missing and a high degree of identity with MAT A-3 can only be found in the first 91 amino acids. Functional conservation between P. anserina and S. macrospora mating-type proteins can be demonstrated by the fact when the entire Smt locus was transformed into a mat+ or mat- nucleus, self-mating was observed (Pbggeler et al, 1997). However, the perithecia produced by the transformants do not contain any asci or ascospores. The formation of barren perithecia in the transformants is dependent on the Smt-al and Smt-Al genes, since each alone can induce the mating reaction in mat- and mat+ strain respectively; fruiting bodies cannot be detected in either mat+ or mat- strains after transformation of plasmid containing SmtA-2 and SmtA-3 only.  Although it is not necessary for a nuclear recognition system to function in 5. macrospora, the mating nuclei are still differentially recognized (Esser & Straub, 1958; Heslot, 1958). It is not clear how differentiation can be accomplished in a population of a single type of nuclei, but mechanisms involving differential expression of mating-types and/or imprinting through male/female origin have been suggested (Coppin et al, 1997).  1.3.7 Ustilago maydis Ustilago maydis (DC.) Corda (or corn smut fungus) belongs to the basidiomycetes, a class of fungi that include many common mushrooms and plant pathogens, such as smuts and rusts. U. maydis is dimorphic: the non-pathogenic, unicellular haploid form and the pathogenic 28  filamentous dikaryotic form (Banuett, 1992). The haploid, yeast-like sporidia grow vegetatively by budding. When sporidia of opposite mating-type are mixed, the formation of irregularly shaped conjugation tubes is induced. The conjugation tubes then fuse and result in the formation of a dikaryon, which is characterized by its long regularly shaped filament. The growth of the pathogenic dikaryon is strictly dependent on the corn plant. The hyphal cells grow within the plant and cause the formation of tumors. The fungal hyphae differentiate within these tumors, where karyogamy takes place, resulting in the formation of diploid spores, teliospores. Upon release from the tumors, the teliospores are spread by the wind. The teliospores germinate on a plant or on a rich medium and produce a short filament, into which the diploid nucleus migrates and undergoes meiosis. A short septated filament (probasidium) that consists of 4 haploid cells is formed (c.f. unicellular basidium in homobasidiomycetes). Each of the haploid cells can produce numerous sporidia by successive budding.  Mating-type locus organization in Ustilago maydis The life cycle of U. maydis is governed by two mating-type loci, a and b (Bolker et al, 1992; Gillissen et al, 1992; Kamper et al, 1994). For the formation of the pathogenic filamentous dikaryon, the haploid mating partners must have different alleles at both loci. The a locus controls fusion events (Trueheart & Herskowitz, 1992) and has two alleles, al and a2. The a locus encodes two structural components of the pheromone response pathway: a pheromone and a pheromone receptor (Bolker et al, 1992; Snetselaar et al, 1996). Two putative pheromone structural genes found in the a locus, mfal (from al) and mfa.2 (from al), encode short polypeptides (of 40 and 38 aa, respectively) which contain a CAAX motif (a motif found in other fungal pheromones; C = cysteine, A = aliphatic amino acid, and X = any amino acid) at the Cterminus. Two other genes that resemble pheromone receptors, pral and pra.2, are found in al and a2, respectively. These two genes encode protein with several putative trans-membrane domains that show similarity to the a-factor receptor (STE3) of S. cerevisiae. The a2 locus contains two additional genes, lga2 and rga.2; although they have unknown functions, their 29  expression is activated by the pheromone response pathway (Urban et al, 1996a, b). Expression of all genes from the a and b loci is enhanced (10 to 50 fold) by the pheromone response pathway through the pheromone response element (PRE, ACAAAGGGA) and the HMG binding protein, PRF1 (Urban 1996b, Hartmann et al, 1996).  The a locus, together with the b locus, controls filamentous growth of the dikaryon: a diploid strain homozygous for a (but heterozygous for b) grows yeast-like unless externally supplied pheromones of the opposite mating type are present (Banuett & Herskowitz, 1989; Spellig et al, 1994; Kamper et al, 1994). A model explaining linkage of the a locus with filamentous growth is as follow: U. maydis sporidia produce a low level of pheromone that is sufficient for mating. Once the pheromone response pathway is activated through receptor binding,/?/// is induced by a post-transcriptional mechanisms (Hartmann et al, 1999). PRF1 in turn induces production of bE and bW (see below), which form heterodimers and bind to genes that promote dikaryotic filamentous growth and sexual differentiation.  A Get protein in the cAMP signaling pathway has been isolated: gpa3 mutants fail to respond to pheromone and are mating-deficient (Regenfelder et al, 1997; Kruger et al, 1998). A gene homologous to yeast MCM1 (see section 1.3.1), umel, is not essential for pathogenic development - it only functions to modulate (increase) the expression of several pheromoneinducible genes (Kruger et al, 1997). Another gene potentially involved in the pheromone response pathway is fuz7 (Banuett & Herskowitz, 1994; Banuett, 1995). It is a homolog of the STE7 MAPKK of S. cerevisiae and is required for a-locus-dependent processes (conjugation tube formation, filament formation, and maintenance of filamentous growth). The signal transduction pathway for pheromone response is apparently conserved between U. maydis and U. hordei (Bakkeren & Kronstad, 1996), which is a bipolar-mating smut (Bakkeren & Kronstad, 1994; Lee et al, 1999).  30  Development after cell fusion is controlled by the b locus (Kronstad & Leong, 1989, 1990; Schulz et al., 1990). The b locus of U. maydis is mutliallelic: at least 25 b alleles exist in nature, implying that there are at least 300 different b combinations that trigger development while 25 combinations do not. Any combination of 2 different alleles triggers development to a similar degree. Heterozygosity at the b locus is required for tumor development, sexual development (meiosis and sporulation) and filamentous growth. This can be demonstrated by the observation that a diploid strain with genotype alalblbl pathogenic, while a diploid with genotype alalblb2  grows yeast-like and is non-  also grows yeast-like but is able to induce  tumors and to form basidiospores. The b locus contains two dissimilar genes, bE and bW, which encode polypeptides of 410 and 626 aa, respectively (Gillissen et ah, 1992). Each b allele contains such a gene pair, e.g., bl allele contains bEl and bWl while b2 allele contains bE2 and bW2. Both genes contain a constant and variable region (Kronstad & Leong, 1990). The constant region is highly conserved (>90%) among the b alleles. The bE polypeptide contains a HD1 homeodomain (WFINAR) whereas the bW polypeptide contains a HD2 homeodomain (WFQNRR) (Kiies & Casselton, 1992a, b). The non-self recognition can be explained by the fact that bE and bW proteins will dimerize (through the N-terminal variable domains) only if they are derived from different alleles (Kamper et al., 1995). In U. maydis, both homeodomains are required for function of the bE/bW complex (Schlesinger et al., 1997), whereas in Schizophyllum commune (another basidomycete; see section 1.3.8), only one of the paired HD1/HD2 complex encodes a homeodomain essential for Aa-regulated development (Luo et al., 1994). The active bE/bW complex is likely to act as a repressor of a repressor, since a mutation in rtfl bypasses the requirement for heterozygosity at b and confers pathogenicity to a mutant haploid strain (Banuett, 1991). Allele specificity of bE and bW has been demonstrated to lie within the N-terminal variable region (Dahl et al, 1991; Yee & Kronstad, 1993, 1998). Interestingly, novel specificity of bE and bW can be created by chimeric construction between two b alleles (Yee & Kronstad, 1993, 1998). It is noteworthy that a single point mutation into the variable region of bE3 leads to a change of specificity (Kamper et al., 1994, 1995). 31  Downstream targets directly regulated by the &-locus have not been identified, although a gene that is involved in filament growth and virulence has been characterized (Giasson & Kronstad, 1995; Laity et al, 1995). The cAMP signaling pathway responsible for budding/filamentous growth regulation is apparently linked with the mating MAPK pathway, since mutants defective in the cAMP pathway are attenuated for the mating and/or virulence function (Gold et al., 1994, 1997; Durrenberger et al., 1998; Durrenberger & Kronstad, 1999). The two pathways share the same Gcc protein (Regenfelder et al., 1997; Kahmann & Basse, 1997).  1.3.8 Coprinus cinereus and Schizophyllum commune Coprinus cinereus (SchaeffenFr.) Gray has two distinct mycelial stages: the uninucleate monokaryon with abundant oidia (uninucleate asexual spore) and the binucleate dikaryon with characteristic clamp connections between each cell (Casselton & Kiies, 1994). Two multiallelic mating-type factors, A and B, determine mating compatibility in C. cinereus. A and B independently regulate different developmental steps. Following fusion between matingcompatible hyphal cells, there is a reciprocal exchange of nuclei (Casselton, 1978). Migration of the donor nucleus triggers the breakdown of the dolipore (septium), a structure which normally precludes passage of nuclei (Giesy & Day, 1965). Once the tip cells have two different nuclei, a clamp cell develops on the side of the tip cell, one nucleus moves into this, then both nuclei divide synchronously. New septa are laid down, cutting one nucleus off in the clamp cell and one in the subterminal cell. The clamp cell then fuses with the subterminal cell and its nucleus travels into this cell. Given the right temperature and light condition the dikaryon will differentiate into mushroom fruiting bodies, in which nuclear fusion, meiosis and sporulation take place (Tymon et al, 1992). The A factor represses asexual sporulation and controls the formation of the clamp cell, nuclear pairing and synchronized nuclear divisions (Raper, 1966; Tymon et al, 1992; Kertesz-Chaloupkova et al, 1998; Kiies et al, 1998), whereas the B factor controls clamp cell fusion and all the nuclear migration steps (Raper, 1966; O'Shea et al, 1998). Both A and B factors are required for maintenance of the dikaryon. 32  The A genes C. cinereus are divided into two functionally redundant subcomplexes, a and P, which are separated from each other by a 7.0 kb-long "homologous hole" (a non-coding region presents in all A factors) (Kiies et al, 1992; Pardo et al, 1996; Luken et al, 1996). Since difference in one subcomplex is sufficient for mating, the estimated number of A specificities are around 160 (Whitehouse, 1949; Raper, 1966). Three types of genes can be found within the A factor. The a-fg (encodes a metallo-endopeptidase) and  (unknown function) genes are not  involved in the A specificity. The rest are the A-specificity gene pairs of homeodomain proteins (al/a2 in Aa; bl/b2 and dl/d2 in Afi; Pardo et al, 1996). The gene pairs are redundant in function and a difference in the alleles of one gene pair, e.g., al-2 and a2-l (the "1" in al-2 means it contains a HD1 domain while the "2" in al-2 indicates that it is allele 2 of the a gene pair; similarly, a2-l is allele 1 of the HD2-containing al gene), is sufficient to generate compatible A factors. The situation here is similar to that of U. maydis where a compatible interaction is triggered by a non-allelic HD1-HD2 protein interaction (Kiies et al, 1994a, c). The requirement for HD1/HD2 heterodimerization for A compatibility is shown in a selfcompatible mutant (constitutive for clamp cell development), in which most of the A locus has been deleted, giving a fused HD2/HD1 chimeric protein which acts as a heterodimer (Kiies et al, 1994b). Because the specificity gene pairs are redundant, it is common to find that some are missing (Casselton & Kiies, 1994; Casselton & Olesnicky, 1998). However, the specificity sequences (such as bl alleles) of the A locus are under tight balancing selection for allele variability (May et al, 1999). Phylogenetic studies show that the N-terminal domain exhibits hypervariability (as required for non-self recognition) while recombination events have homogenized the C-terminal region to preserve functional domains (Badrane & May, 1999). These data explained how bl alleles have evolved high levels of variation while at the same time conserved function.  Two coiled-coil dimerization domains are found in HD1 proteins (Gieser & May, 1994; Banham et al, 1995). The fact that the relative positions of the dimerization domains are 33  different among a, b and d HD proteins gives an explanation why proteins from different subfamilies do not cross-react (Padro et al, 1996). The dimerization of HD1/HD2 proteins is important because the HD1 protein provides the nuclear localization and activation domain while HD2 protein provides the homeodomain required for DNA-binding (Asante-Owusu et al, 1996; Spit et al, 1998).  The B mating type (required for nuclear migration during somatic and clamp cell fusion) of C. cinereus is defined by a single locus, with three sets of functionally redundant, yet independent sub-family of genes (O'Shea et al, 1998). Each sub-set contains a 7transmembrane helix receptor gene (rcb) and two CAAX-modified pheromone precursor genes (phb). Like the A locus, only one subfamily within the B locus has to be different for mating compatibility. C. cinereus pheromones genes from a particular sub-family that can only react with receptors from a different allele of the same sub-family. The mechanism for such pheromone-recognition discrimination is still unknown, although the secondary and tertiary structures of the receptors may play a role in the recognition process (Casselton & Olesnicky, 1998). What is known is that the receptor protein is G-protein-coupled; a single amino acid change in the transmembrane domain VI of one receptor is constitutively activating and confers a self-compatible mating phenotype (Olesnicky et al, 1999). So like JJ. maydis, a pheromone response pathway is required for G-protein-mediated signal transduction. However, unlike U. maydis, mating-type gene expression is not dependent on pheromone signaling (Richardson et al, 1993). Although pheromone signaling is also not required for mate attraction (c.f. U. maydis; Snetselaar et al, 1996), B mating-type genes are required for clamp cell fusion, a process mimicking fusion of haploid sporidia (Casselton & Olesnicky, 1998). Components downstream of the mating cascade remain to be identified in C. cinereus, however, an HMG-box encoding gene affecting sexual morphogenesis (which is regulated by both A and B genes), peel, has been characterized (Murata et al, 1998).  34  The A mating-type system of Schizophyllum commune Fr. is similar to that of C. cinereus (Casselton & Kiies, 1994) except that the Aa and A/3 complex is further apart (8 map units; Novotny et al., 1991). Analysis of 114 isolates identified 96 A alleles and 56 B alleles (Raper et al, 1958). Genes required for fruiting are regulated by the A and B factors (Wessels, 1992; Schuren et al, 1993). Two genes encoding homeodomain proteins, y and z, are found in the Aa locus (Stankis et al., 1992; Specht e al., 1992). Active Y-Z heterodimers can only be formed between alleles from different mating-types (Magae et al., 1995), which in turns act as a transcriptional factor for Aa-regulated developmental genes (with the Y homeobox as the active motif; Luo et al., 1994). The A/3 locus, which is functionally redundant to the Aa locus, is partially characterized and encodes a gene product (V) with a homeodomain similar to those found in Y and Z (Shen et al, 1996).  The B mating-type genes are defined by two discrete loci, Ba and Bfi (Parag & Koltin, 1971; Specht, 1995). The two loci contain independent but redundant genes (recombination between two loci does not lead to self-compatibility). The Ba and 5/3 loci (Wendland et al, 1995; Vaillancourt et al, 1997) contains independent subfamilies of gene cassettes, with one receptor {bar or bbr) and three pheromone genes (bap( 1-3) or bbp(l-3)).  The pheromone genes  contain farnesylation signal CAAX as found in yeast a-factor. The pheromone receptors have sequence similarity to other pheromone receptors such as STE3 from yeast and prallpra2 from U. maydis. A single pheromone can activate more than one receptor and one receptor can recognize more than one pheromone (Wendland et al, 1995). Nine Ba and nine 5/3 alleles have been found in nature and eight of the pheromone genes have been analyzed (Kothe, 1999; Hegner et al, 1999). With nine Aa and 32 A/3 alleles, 23,328 different mating-types are possible (Kothe, 1999). The pheromone/receptor system may give rise to nuclear identity (by nuclearlimited expression) that facilitates the process of dikaryon proliferation and induction/ continuation of nuclear migration throughout sexual development (Schuurs et al, 1999;  35  Raudaskoski, 1998). A gene that is controlled by the 5-regulated pheromone response system and another gene induced by dikaryon proliferation have been isolated (Lengeler & Kothe, 1999a, b). The pheromone response pathway of 5. commune is similar to those from yeast, since the pheromones/receptors of Schizophyllum can induce the yeast pheromone response pathway through the yeast G protein (Fowler et al., 1999).  1.3.9 Other filamentous ascomycetes Magnaporthe grisea The rice blast pyrenomycete Magnaporthe grisea contains idiomorphs of 2.5 kb and 3.5 kb ( M A T 1 - 2 ) . introduction of the M  AM A T 1 - 1  A T 1 - 1  ( M A T 1 - 1 )  clone was isolated by subtractive hybridization. The  clone into M  A T 1 - 2  recipients results in self- and dual-maters (Kang  et al., 1994). Dual-maters containing both mating-type genes are unstable and dual-mating ability is easily lost through a cross or even vegetative growth. M. grisea field isolates are poor maters (generally female-sterile and poorly male-fertile) (Valent et al., 1986), suggesting that mating is not important in pathogenesis. However, mating behaviour has been linked with appressorium (infection structure) by G-proteins (Liu & Dean, 1997). Why M. grisea mate poorly in the field is still unknown, but it is unlikely to be due to a limitation in pheromone precursor expression (Shen et al., 1999).  Pyrenopeziza brassicae In the mat A-type idiomorph from heterothallic discomycete Pyrenopeziza brassicae, a metallothionein-like protein is encoded by the mating-type locus in addition to mat A-l and mat A-3-like proteins (metallothioneins sequester metal and may function in oxygen detoxification and sulphur metabolism); a matA-2 homolog is not present (Singh & Ashby, 1998). It is not clear how a metal-sequestering protein may be involved in mating, but it was suggested that the metallothionein could be an environmental sensor for metal ion concentration in plant and subsequently trigger sexual morphogenesis. Alternatively, it could provide a nuclear recognition 36  mechanism since it is only found in MAT 1-2 and metallothionein mRNAs are shown to localize in the perinuclear cytoplasm and cytoskeletal-bound polysomes (cited in Singh & Ashby, 1998). PCR and hybridization data showed intra-specific conservation of mating-type genes (including the metallothionein gene) between P. brassicae and closely related Tapesia yallundae (Singh et al, 1999).  Glomerella cingulata Most heterothallic ascomycetes contain a single mating-type locus with two alternate alleles (so-called "bipolar or biallelic mating system"). By contrast, most basidiomycetes have a "tetrapolar mating system" in which multiple alleles exist for each of two mating-type loci (Casselton & Olesnicky, 1998). Multiple mating-types infilamentousascomycetes is only reported in the pyrenomycete Glomerella cingulata, where there are at least three mating-type groups (MATA-1 to MATA-3) that are self-sterile but cross-fertile (Cisar & TeBeest, 1999).  Chromocrea spinulosa Unlike yeast (Haber, 1998), mating-type infilamentousascomycetes is believed to be stable and mating type switching (transposition of a silent cassette of mating-type alleles into the active mating type locus) does not happen frequently. However, evidence for unidirectional switching of mating-type has been reported in filamentous ascomycetes (Perkins, 1987), such as Chromocrea spinulosa (Mathieson, 1952), Sclerotinia trifolium (Uhm & Fujii, 1983), Botrytinia fuckeliana (Faretra & Pollastro, 1996), Ceratocystis coerulescens (Harrington & McNew, 1997) and Glomerella cingulata (Wheeler, 1950). C. spinulosa is apparently selffertile, but in its asci, four progeny are self-fertile and four are self-sterile. The self-fertile progeny always produce four self-sterile and four self-fertile progeny; the self-sterile isolates will cross with self-fertile isolates. The self-fertile progeny contain sequences for MAT-1 (cc-domain protein) and MAT-2 (HMG protein) homologs, but the self-sterile progeny contain sequences only for MAT-1 protein (G. Turgeon, personal communication). How a single culture produces 37  progeny with different mat gene organization is unclear, but a programmed genomic rearrangement comparable to mating type switching in yeast is possible.  1.4 HETEROKARYON  INCOMPATIBILITY  IN  FUNGI  1.4.1 Introduction to heterokaryon incompatibility When a spore of a filamentous fungus germinates, hyphae (filament-shaped multinucleate cells) grow by hyphal tip extension and branching. Hyphal tip fusions are continuously formed (a process called anastomosis), giving a network of hyphae called a mycelium, in which the fungus grows and propagates. Occasionally, when two fungal individuals meet, they may undergo hyphal fusion and form a heterokaryon (an individual that contains genetically distinct nuclei within the same cytoplasm). However, a viable, stable heterokaryon between two isolates can only be formed if they contain compatible alleles at genetic loci that restrict heterokaryon viability. These loci are called het (Zieterokaryon) or vie (vegetative mcompatiblity) loci. Genetic differences at any of these het loci can result in inhibited growth of the heterokaryotic cells and compartmentalized cell death by a lytic process (Garnjobst & Wilson, 1956). This growth inhibition/cell death phenomenon is termed heterokaryon incompatibility (and sometimes referred to as heterogenic, vegetative, or somatic incompatibility). In ascomycetes such as N. crassa, heterokaryon incompatibility is strictly a vegetative feature; differences in one or more het loci do not result in an incompatibility reaction between mating nuclei during the sexual cycle and do not impair fertility (Begueret et al., 1994).  Heterokaryon incompatibility is thought to be a universal phenomenon among filamentous ascomycetes and basidiomycetes (reviewed by Glass & Kuldau, 1992; Leslie, 1993; Esser & Blaich, 1994; Begueret et al, 1994; Worrall, 1997; Glass et al, 2000). In N. crassa, heterokaryon formation by hyphal fusion is virtually excluded in nature by the action of het genes (Mylyk, 1976a; Perkins & Turner, 1988; Pandit & Maheshwari, 1996). The  phenomenon  38  of heterokaryon incompatibility is thought to limit the spread of deleterious organelles and infectious elements in fungal populations (Caten, 1972). Experimental data supports this theory - cytoplasmic transfer of virus and detrimental plasmids within fungal populations is reduced or completely blocked by het actions (Anagnostakis, 1982a; Debets et al., 1994; Van Diepeningen et al., 1997, 1998). Heterokaryon incompatibility may provide a selective advantage for favorable mutations by preventing exploitation of heterokaryons by non-adaptive nuclei (Haiti et al., 1975), and by protecting unfertilized cultures from looting of maternal resources during sexual reproduction (Debets & Griffiths, 1998). Others have proposed that the presence of het genes would limit out-breeding and thus favoring evolution of a single group leading to speciation (Esser & Blaich, 1973, 1994).  The phenomenon of heterokaryon incompatibility can be demonstrated in several ways. For example, (i) the failure for two strains to form a stable heterokaryon with continuous growth (Garnjobst, 1953), (ii) microscopic (light and electronic) features of protoplasmic killing (Garnjobst & Wilson, 1956; Jacobson et al., 1998) and (iii) the formation of a barrage at the point of confrontation of incompatible strains (Reinhardt, 1892). The phenotype of the barrage varies between different fungal species, from a pigmented line of dying hyphae to a clear zone of self-lysis (Esser & Blaich, 1994). Heterokaryon incompatibility loci can be identified using forced heterokaryon formation between near isogenic strains (Garnjobst & Wilson, 1956; Pittinger & Browner, 1961). Alternatively, they can be identified by abnormal growth and morphology of fungal strains with partial diploid heterozygous for a het gene (Newmeyer & Taylor, 1967). Most of the het loci in Neurospora have been identified using partial aneuploids or disomic methods, in which progeny with partial diploids are generated by crossing a translocation strain with a wild-type strain (Perkins, 1975; Mylyk, 1975).  Genetic studies in several ascomycetes show that there are a number of het loci in each species, for examples, there are at least eleven in N. crassa, nine in P. anserina, eight in  Aspergillus nidulans, and six in Cryphonectria parasitica (Begueret et al., 1994). Several het genes and suppressors of incompatibility have been cloned from N. crassa and P. anserina, while heterokaryon incompatibility in A. nidulans (Dales & Croft, 1990; Anwar et al., 1993) and in C. parasitica (Cortesi & Milgroom, 1998; Milgroom & Cortesi, 1999) has been only studied genetically. In this chapter, the characteristics and nature of het loci and their suppressors/ modifier genes in N. crassa and P. anserina are discussed.  1.4.2 N. crassa mat locus (The mating function of the N. crassa mat locus is summarized in section 1.3.3) Entry into the sexual cycle in filamentous ascomycetes requires the fusion of reproductive structures of opposite mating-types. The mat A (A-l, A-2 and A-3) and mat a gene (a-l) genes regulate fusion and post-fertilization events during sexual development (see section 1.3 for review of mating-type). In iV. crassa (but not in all filamentous ascomycetes), the mat locus also functions as a het locus (Beadle & Coonradt, 1944; Garnjobst, 1953). The fusion of mat A and mat a hyphae during vegetative growth results in growth inhibition, hyphal compartmentation and death (Glass & Kuldau, 1992); the mating-type incompatible heterokaryon is aconidiating, slow-growing (10-20 fold slower than a compatible heterokaryon) and contains compartmentalized cell death events similar to those observed in het-c and het-6 incompatibility reactions (see section 1.4.3). In most cases, the incompatibility function of mat was found to be inseparable from its sexual function, indicating that either the two functions are conferred by the same gene, or they are encoded by separate genes that are closely linked (Newmeyer et al, 1973; Griffiths, 1982). An exceptional a mutant, a , is fertile but heterokaryon compatible with A m3S  strains, suggesting the mating and incompatibility functions are separable (Griffiths & DeLange, 1978). The mating-type associated heterokaryon incompatibility is due to the molecular action of MAT A - l and MAT a-l, since mutations that affect both mating and incompatibility functions are mapped in mat A-l and mat a-l genes (Glass et al., 1990; Staben & Yanofsky, 1990). Moreover, introduction of mat A-l into a recipients (or the introduction of mat a-l into A 40  recipients) is sufficient to trigger heterokaryon incompatibility (Glass et al, 1990; Staben & Yanofsky, 1990). Additional mat A-l and mat a-1 mutants that separate mating and heterokaryon incompatibility have been obtained, indicating that the two functions lie in separate functional domains of the protein and are not always mutually inclusive (Staben & Yanofsky, 1990; Philley & Staben, 1994; Saupe et al, 1996b).  Partial diploid strains that contain a duplication for mating type show inhibited growth initially (a phenotype called Dark Agar; Newmeyer, 1968) and escape to a wild-type growth rate after a few days; duplication of mating-type can be obtained by crosses heterozygous for inversion In(ILR)H4250 (Newmeyer & Taylor, 1967). The escape strains usually contain a mutation in either the A or a locus. However, a mutation unlinked to the mating-type locus, tol (to/erant), suppresses mating-type associated heterokaryon incompatibility (Newmeyer, 1970). Attempts to isolate additional suppressors of mar-mediated incompatibility results only in the isolation of additional tol alleles (Vellani et al, 1994).  Mutations in tol are recessive such that tol A and tol a strains form a vigorous heterokaryon (DeLange & Griffiths, 1975). tol maps close to trp-4 in linkage group IV and does not suppress other heterokaryon incompatibility systems (Newmeyer, 1970; Leslie & Yamashiro, 1997). Other than the ability to suppress maf-mediated incompatibility, tol mutants show a wildtype phenotype during vegetative growth. Mutation in tol does not affect mating, however, the tol mutation (when homozygous) has been shown to restore fertility in the fmf-1 (/emale and male/ertility) mutant (Johnson, 1979), suggesting tol may play a role during sexual cycle.  Why MAT A - l and MAT a-1 exhibit such diametrically opposed functions in the sexual and vegetative phases in N. crassa and their relationship to each other is still an unsolved enigma of mating type function. The focus of this thesis is to study the heterokaryon incompatibility as  41  mediated by mat. Extensive reviews on mating-type-associated incompatibility are provided in later chapters.  1.4.3 N. crassa het-c locus The het-c locus is the best-studied het locus in N. crassa. The het-c locus was first identified in forced heterokaryons of near isogenic strains that differed in het-c (Garnjobst, 1953) and later characterized using translocation strains duplicated at het-c locus (Perkins, 1975). Although not as strong as the het-6 and mat incompatibility reactions, the incompatibile het-c heterokaryon displays a distinctive phenotype: aconidiating, slow-growing mycelium with curly hyphae. The het-c allele was isolated by a chromosome walk from a closely linked gene, pyr-4 OR  (Saupe et al, 1996a). The het-c activity was localized in overlapping regions of two cosmids: OR  introduction of either cosmid into a het-c strain led to growth-inhibited transformants PA  resembling het-c incompatible heterokaryons or partial diploids.  The 966-amino acid HET-C  0R  polypeptide contains a putative signal peptide, a coiled coil  domain and a C-terminal glycine-rich domain (Saupe et al., 1996a). The presence of sequences similar to a signal peptide suggests that HET-C might enter the secretory pathway. The 5-heptad coiled-coil domain could provide a dimerization motif for protein-protein interactions. Glycinerich domains are found in a large number of proteins such as extracellular or cell envelope proteins, RNA-binding proteins and keratin. These domains are glycine and serine-rich and contain regularly spaced aromatic residues that are proposed to allow tension-adaptable proteinprotein interactions that provide extensibility and elasticity (Steinert et al., 1990). Several het-c mutants have been obtained via RIP (repeat-induced point) mutation (Saupe et al, 1996a). They no longer mediate het-c vegetative incompatibility (i.e., they are heterokaryon compatible with all het-c alleles) but are otherwise normal in vegetative growth and sexual reproduction.  42  Genetic and molecular characterization of the het-c gene from two other N. crassa strains (het-c and het-c ) demonstrated that the het-c locus is functionally multi-allelic (Saupe & PA  GR  Glass, 1997). Transformation results agree with previous genetic data that het-c incompatibility is' mediated by at least three alleles (Howlett et al., 1993). Multi-allelism is rare in allelic incompatibility systems and has only been reported in one other case (Dales et al., 1993). DNA sequence analysis of the three het-c alleles revealed a highly variable domain of 34-48 amino acids (position 247 to 284 in HET-c ; Saupe & Glass, 1997). This highly variable domain, OR  which includes substitutions and insertion/deletions (including a 5-aa HET-c -specific and a 15OR  aa HET-c -specific insertion), determines het-c allelic specificity. Swapping of this highly PA  variable specificity domain effectively changes the specificity of a het-c construct as tested by transformation assays (Saupe & Glass, 1997). Recent experiments on artificial het-c constructs show that novel specificity can be obtained by changing deletion/insertion pattern of the variable domain (J. Wu & N.L. Glass, unpublished results). These data show that the spacing (and not so much amino-acids composition) within the het-c variable domain is crucial in determining het-c specificity.  Evidence for balancing selection at the het-c locus was shown by studies of the evolutionary pattern of het-c in related species and genera to N. crassa (Wu et al., 1998b). The HET-c specificity region of 40 different isolates, representing 11 different species and three genera within the Sordariaceae (Neurospora, Gelasinospora and Sordaria), was shown to exhibit trans-species polymorphisms, i.e. DNA sequences of the het-c specificity region fell into groups by het-c allelic type rather than according to species. These data show that allelic specificities at het-c were generated in an ancestral species and have been maintained over 35 million years and during multiple speciation events. Moreover, the specificity domain of het-c also exhibits an excess of nonsynonymous substitution over synonymous substitutions, suggesting a selection to maintain diversity at this locus (Wu et al., 1998b). The het-c locus thus shares the evolutionary characteristics with other non-self recognition systems such as the MHC (major 43  histocompatibility complex; Ayala et al, 1994) loci in mammals and the S (self-incompatibility; Ioerger et al., 1990) locus in plants. The het-c locus was found to be polymorphic with all three alleles showing near equal frequency in a population isolated from a 5-hectare sugarcane field in Louisiana (Wu et al., 1998b). These data suggest that the non-self recognition function mediated by difference at het-c is of biological significance in N. crassa.  Early studies by Wilson and co-workers (1961) showed that micro-injection of cytoplasm from a donor cell can kill a recipient of an incompatible het-c type. The killing factor in the cytoplasmic extracts can be inactivated by heat (60°C) or proteases (but not by nucleases), suggesting the incompatibility factor involved was a protein (Wilson et al., 1961; Williams & Wilson, 1966). The incompatible component transferred is likely to be HET-C itself. Recent coimmunoprecipitation experiments using GFP (green fluorescent protein) and HA (hemagglutinin) tagging show that HET-c homodimerization of HET-c  OR  OR  and HET-c  PA  can be co-precipitated while  was not detected (J. Wu & N.L. Glass, unpublished results).  These data showed that molecular recognition mediated by het-c is due to dimerization of unlike HET-C proteins.  Attempts to isolate suppressors for het-c incompatibility has been made by Arganoza and co-workers (1994). However, the spontaneous and insertional inactivation mutants that tolerate het-c difference have not been further investigated. Another suppressor (vib-1) of het-c heterokaryon incompatibility has been isolated (Q. Xiang & N.L. Glass, unpublished results). Other than the ability to tolerate het-c differences, the phenotype of vib-1 (vegetative incompatibility Mocked) include poor aerial hyphae growth, female-sterility and hyphal fusion defects.  Microscopic and ultrastructural features associated with het-c (and het-6) incompatibility have been examined (Jacobson et al., 1998). Morphological features of partial diploids 44  heterozygous at the het-c locus, as revealed by transmission electron microscopy, include nucleolar release, cell shrinkage, extensive organelle degradation and plasmolysis. These ultrastructural changes in dying cells are similar to the features of apoptosis (programmed cell death; Konopleva et al, 1999) in higher eukaryotes. DNA fragmentation, a hallmark feature of early apoptosis as mediated by Ca -dependent endoclease cleavage of nucleosomes, is also 2+  observed by TUNEL (terminal deoxyribonucleotidyl transferase) assay on /ier-c-incompatible hyphal fusion cells (R. Bostock & S. Marek, unpublished results). These microscopic and biochemical experiments suggest that fungal vegetative incompatibility and apoptosis in plants and animals may share common biochemical and genetic mechanisms. It is noteworthy that an apoptosis-like form of programmed cell death has been reported in filamentous fungi (Roze & Linz, 1998).  Homologs of het-c have been found in P. anserina and A. nidulans. The Podospora het-c homolog (hch) has high sequence similarity to N. crassa het-c, although the specificity region is divergent (Saupe et al, 2000). Population studies show that hch is apparently non-polymorphic in P. anserina and therefore may not act as a het locus. It is possible that the occurrence of het-c polymorphism and the acquisition of het-c as a het locus occurred after speciation between Podospora and Neurospora, 75 millions years ago. However, hch appears to be potentially active and could act as a het gene in P. anserina if polymorphism exists - heterologous expression of any of the three N. crassa het-c alleles in P. anserina led to growth defect and cell death that mirrored N. crassa het-c incompatibility (Saupe et al., 2000). The Aspergillus het-c homolog (nipQ was isolated from a yeast 2-hybrid screen using nim-A as bait. NIMA (never in meiosis) is a cell cycle regulator that is responsible for the transition from interphase to prophase (G2-M) (Osmani et al, 1988). NIMA, a SER/THR protein kinase, is proposed to target G2/M checkpoint controls by promoting nuclear localization of CDCl/cyclin B (Wu et al, 1998c). Over-expression of NIPC causes cell cycle arrest at interphase, suggesting that the het-c homolog may act as a negative regulator of cell cycle (S. Osmani, unpublished results). It is not clear if 45  nipC is polymorphic in nature and acts as a het gene in A. nidulans. Polymorphism is found at het-c in a pseudohomothallic species of Neurospora, N. tetrasperma (Powell et al., 2000). These data suggested the self-fertile fungal species, thought to be non-outcrossing in nature, does outcross. The fact that diversity is found at het-c locus while other genes are highly homozygous (Merino et al., 1996) illustrates that non-self recognition may also be selected for in a fungal species that is effectively homothallic (Powell et al., 2000).  1.4.4 N. crassa het-6 locus het-6 was first identified by partial diploid analysis - strains carrying partial diploid at the het-6 locus are inhibited in their growth (Mylyk, 1975). The region of translocated (duplicated) DNA, T(IIL—>IIIR)AR18 defines the het-6 locus. The het-6 locus was later mapped to a 35 kb region within the translocated fragment, which is the amount of deletion at one of the het-6 loci required for the partial diploid strain to "escape" from the incompatible phenotype (Smith et al., 1996). The incompatibility phenotype of het-6 is among the most severe - the heterokaryon has a 100-fold reduced growth rate (Perkins, 1975; Jacobson et al., 1998).  Cosmids spanning the het-6 translocation region were isolated by probing the genomic library with L G II DNA from wild type and T(1IL->IIIR)AR18 DNA (Smith & Glass, 1996; Smith et al., 1996). Four of the six cosmids isolated by this method were used to initiate a chromosome walk, resulting in a near-contiguous segment across the het-6 translocated region (Smith et al., 1996). Two of the cosmids gave a dramatically reduced number of transformants (20 fold less) when introduced into strains that have a different het-6 haplotype, suggesting that het-6 function is within the overlapping region (Smith et al., 2000b). Two genes (14 kb apart) identified within this region, named het-6 and un-24, exhibited incompatibility activity as tested by the transformation reduction assay and were further characterized (Smith et ah, 2000b).  46  The un-24 gene encodes the large subunit of ribonucleotide reductase (RNR) (Smith et al., 2000a). The class I ribonuclease reductase is essential for de novo synthesis of DNA, replacing the C-2' hydroxyl of ribonucleoside diphosphate (NDP) with a hydrogen to give deoxyribonucleoside diphosphate (dNDP) (Jordan & Reichard, 1998). A temperature-sensitive mutant (un-24 ) grows at a wild-type growth rate below 30°C but does not grow when the TS  temperature exceeds 34°C (Smith et al, 2000a). The un-24 allele has three GC—»AT transition TS  mutations, one of which changes a conserved amino-acid (C26—»Y) located within the allosteric activity site where ATP (activator) or dATP (inhibitor) binds. The mutations in un-24 , which TS  may affect proper folding of the allosteric activity site at elevated temperature, can be suppressed by osmotic remediation (which reduce water content and change the conformation of enzymes). Ribonucleotide reductase activity is apparently separable from incompatibility activity within un24. When DNA from the un-24 allele (derived from wild-type OR background) is transformed TS  into spheroplasts of alternate haplotype (PA background), a 20-fold reduction in transformation frequency is observed even at restrictive temperature (37°C) (Smith et al., 2000b). A C-terminal domain, which is variable between UN-24 and UN-24 and does not exist in other RNRs, may PA  OR  be responsible for the incompatibility.  The het-6 gene encodes a 680-aa polypeptide (Smith et al., 2000b). Three regions of sequence similarity are found between HET-6  OR  and HET-E, which mediates non-allelic  incompatibility (with HET-C) in P. anserina (Saupe et al., 1995a). The three regions of similarity are distinct from the GTP-binding domain and the 13-transducin-like repeats in HET-E and may thus represent a novel incompatibility domain. The alternative het-6 allele encodes a protein (HET-6 ) that is only 68% identical to HET-6 PA  0R  with polymorphic positions scattered  throughout the ORFs.  Transformation results suggest that un-24 and het-6 genes do not act as allelic incompatibility loci. Although a transformation reduction is observed when un-24 and het-6 OR  OR  Al  DNA were introduced into PA recipients, no incompatibility reaction is triggered when un-24  PA  or het-6 alleles are transformed into OR spheroplasts (Smith et al, 2000b). This result PA  suggests that there are additional factor(s) within the het-6 region (that span both het-6 and un-24 genes) that mediate incompatibility and that incompatibility may be of non-allelic nature.  The het-6 and un-24 polymorphisms are apparently selected for in nature. In a population study of 40 wild N. crassa isolates from Louisiana, OR-like and PA-like alleles at het-6 and un-24 were equally frequent (Mir-Rashed et al., 2000). This result suggests that, like het-c, the het-6/un-24-mediated non-self recognition may also be subject to balancing selection. Interestingly, all the Louisiana isolates are of either het-6 un-24 or het-6 un-24 haplotype OR  OR  PA  PA  (while all combinations of het-6 and het-c were found); the het-6 un-24 or the het-6 un-24 OR  PA  PA  OR  haplotype cannot be identified in 126 wild isolates (Smith et al., 2000b). These data indicate that the het-6-un-24 region is inherited as a block. The recombination block in probably due to lack of recombination between het-6 and un-24, since a low level of DNA similarity is detected in the intergenic region (Mir-Rashed et al., 2000). Examples of recombination block can also be found in other Neurospora regions, such as the spore killer locus (Campbell & Turner, 1987) and the mating-type locus (Gallegos et al, 2000). Taken together, these data suggest that un-24 and het6 act as an incompatibility complex.  1.4.5 P. anserina het-s locus The het-s/S system mediates allelic incompatibility in P. anserina and is the most studied het locus in that species. The incompatible het-s and het-S alleles originated from geographic strains, s and S (from Normandy, France; Rizet, 1952). The two strains were analyzed for their het genotype (Bernet, 1967a) and were backcrossed for 20 generations to give isogenic strains differ only at the s locus. Fusion between strains with differences in het-s leads to death of heterokaryotic cells, as shown by the formation of a barrage (a dense and unpigmented line of dying cells) in the region where the two isolates meet. 48  Cloning of het-s was made possible by the identification of a natural het-s allele, het-s , 1  from strain H collected in Burgundy (Bernet, 1967a). The het-s allele is compatible with both x  het-S and het-s (later characterization of the het-s" allele revealed that it has a duplication of 46 bases, leading to a frameshift mutation and a truncated version of het-s; Deleu et al., 1993). The het-s gene was cloned by transformation of a s" strain with D N A from a s cosmid library; transformants (from pools of cosmids) were screened for the ability to induce barrage formation when confronted with the S strain (Turcq et al., 1990).  Both het-S and het-s genes encode novel polypeptides of 289-aa (Turcq et al., 1991). H E T - S and H E T - s differ by 14 amino acids and are apparently not essential for cell viability. Strains carrying a disrupted copy of the het-S or het-s (named strains S° and s°) behave like wild type in growth, pigmentation, differentiation and fertility (Turcq etal., 1991). D N A Sequence analysis of 4 natural het-s alleles from P. comata revealed that only 8 of the 14 polymorphic amino acids strictly correlate with het-s/S specificity. Further analysis of het-S/s by chimeric constructs and site-directed mutagenesis indicated that a single amino-acid change (H33P) in hetS allele can switch its phenotype to het-s (Deleu et al., 1993).  There are several lines of evidence indicating that the het-s gene product behaves as a prion analog (Coustos et al., 1997). A prion is an abnormal conformational state of a normal cellular protein capable of "infecting" and converting the normal form (PrP ) into the altered c  ones (PrP ) (Prusiner, 1998). A n infectious prion is implicated in a mammalian Sc  neurodegenerative disease called spongiform encephalopathies (e.g. mad cow disease). The normal P r P protein resides predominantly on the surface of the neuron while the infectious, c  harmful form of the prion protein P r P  Sc  is more stable (resistant to protease) and accumulates in  the cytoplasm (Horwich & Weissman, 1997). The two forms of the proteins contain the same chemical structure but have alternative secondary structures that may explain their conformational differences. Infectious propagation of protein conformation has also been  49  reported in fungi (Silar & Daboussi, 1999; Wickner et al., 1999). [URE3] (which derepresses nitrogen catabolism) and [PSI+] (which increases suppression of non-sense mutations) are two non-mendelian yeast elements. They are shown to be the inactive forms of cellular proteins ( U R E 2 and SUP35, respectively) that are capable of converting the normal forms into the inactive forms (Wickner et al., 1995, 1999).  It has been known for decades that a strain carrying the het-s allele can express a neutral [Het-s*] phenotype, which is compatible with both het-s and het-S strains. Such a non-reactive [Het-s*] state (formerly het-s ) can be found in wild-type isolates, i n protoplasts prepared from s  het-s mycelium (Belcour, 1976) or in progeny recovered from a cross between het-S and het-s strains (Rizet & Schecroun, 1959). Self-crosses from het-S and het-s strains yield progeny with uniform phenotypes ([Het-S] and [Het-s], respectively). When the [Het-s] phenotype is present in the male (but not in the female), the [Het-s] phenotype is not transmitted; when a het-s strain (expressing [Het-s] phenotype) acts as female, all het-s progeny have the [Het-s] phenotypes (Beisson-Schecroun, 1962). These data show that other than the het-s genetic composition, a cytoplamic determinant found in the [Het-s] strain is crucial for the transmission of the [Het-s] phenotype. Additionally, the [Het-s*] strain can revert to [Het-s] phenotype spontaneously during vegetative growth (at a low rate of under 10" per nucleus; Beisson-Schecroun, 1962) or 7  through anastomoses (independent of nuclear transmission) with a het-s strain (Rizet & Schecroun, 1959; Beisson-Schecroun, 1962). From these results, it was hypothesized that the H E T - s protein can induce (and regulate) its own expression (Beisson-Schecroun, 1962). However, modern concepts of an infectious protein capable of catalyzing its own modification led to a different interpretation of the results.  Recent observations on the [Het-s]/[Het-s*] phenomenon suggested that the H E T - s protein acts as a prion (Coustos et al., 1997): (i) a functional het-s gene is required for expression of [Het-s] phenotype and for the propagation of transformation from [Het-s*] to [Het-s]; (ii)  50  Over-expression of het-s gene led to an elevated frequency of spontaneous [Het-s*] —> [Het-s] transition; (iii) Interaction between HET-s/S (S/S, s/s, S/s) proteins (monomers and multimers) were detected; (iv) H E T - s and H E T - s * proteins were present i n similar amounts in the respective strains, with similar electrophoretic mobility under denaturing condition; (v) H E T - s is more resistant to proteinase K than H E T - s * ; (vi) the [Het-s*] —> [Het-s] transition is independent of protein translation. A l l of these properties support the prion model of self-propagating conformational modification.  In an effort to isolate intra- and extragenic suppressor for het-s/S incompatibility, a selfincompatible strain containing both antagonistic alleles (brought together by transformation) was UV-mutagenized and escape sectors recovering normal growth rate were isolated (Coustou et al., 1999). O f 44 analyzed sectors, 27 resulted from mutations outside the het-s/S locus while 17 were intragenic suppressors (14 affected het-S and 3 affected het-s). The characterization of the intragenic suppressor mutants plus the additional functional analysis i n het-s (by deletion constructs and site-directed mutagenesis; Coustou et al., 1999) revealed the amino acid positions in H E T - s / S that are crucial in determining reactivity (e.g. H E T - s aa 1-112 confers full [Het-s] functionality), allele specificity (e.g. single amino acid changes at several positions lead to altered specificity), converting activity (e.g. H E T - s aal-25 allows prion propagation) and metastability (e.g. the het-S K 2 0 1 R mutation leads to a metastable [Het-S*] phenotype).  1.4.6 P. anserina het-c/e (het-c/d) system Several loci mediate non-allelic incompatibility in P. anserina.  In non-allelic  incompatibility systems, special combinations at two separate and distinct loci result in incompatibility. In a cross between non-allelic incompatible strains, progeny with the two antagonistic genes are inhibited upon germination and subsequently destroyed in a lytic process (Bernet, 1965; Bernet et al., 1973). The het-c locus in P. anserina is involved i n two non-allelic incompatibility systems, het-c/e and het-c/d (Bernet 1967c, 1992). Each of these three loci are  51  multi-allelic: from genetic analysis of 17 wild-type isolates, 4 alleles are found at het-c (cl to c4) and in het-e (el to e4) while 3 alleles are present in het-d (dl to d3) (Bernet, 1967b). Each het-c allele is incompatible with a specific set of het-d and/or het-e alleles. For example, het-c2 is incompatible with het-el and het-dl while het-c3 is incompatible only with het-e2. The het-c/d and het-c/e systems could be redundant since H E T - E and H E T - D polypeptides are very similar (Barreau et al., 1998; J. Begueret, unpublished results).  The het-c gene was cloned by phenotypic expression of het-c2 allele. Pools of cosmids from a het-c2 library were introduced to a het-c3 strain and transformants were screened for incompatibility with a het-el strain in a barrage test (Saupe et al., 1994). The 208-aa H E T - C 2 polypeptide displays similarity to a glycolipid transfer protein ( G L T P ) isolated from pig brain, which is responsible for transport of specific glycolipids (such as glycosphingolipids and glyceroglycolipids) from donor to acceptor membrane (Abe, 1990). H E T - C 2 contains a putative amphipathic cc-helix, a functional domain that is involved in protein-protein interaction and lipid binding in some proteins. Disruption of het-c by gene replacement generated two mutants with distinct, recessive phenotypes (Saupe et al., 1994). The het-cAA (partially disrupted) strain is colonial, has higher branching of apical hyphae and is more susceptible to growth-inhibitors that target cell-wall biosynthesis (e.g. 2-deoxyglucose). The het-cAB (total-knockout) mutant is not affected in vegetative growth, but is drastically impaired in ascospore production - in a het-c AB x het-cAB cross, over 80% of the perithecia do not contain any mature ascospores while the remaining ones have abortive asci with rare, asynchronously-developing ascospores. The heterogeneity i n ascospore size could be an outcome of defects in the distribution of nuclei. The sequence similarity and the mutant phenotypes suggest that H E T - C could be involved in maintaining normal membrane composition and vesicle transport.  To delineate the molecular basis of het-c allelic specificity, three more wild-type het-c alleles and two mutant alleles have been characterized (Saupe et al., 1995b). Sixteen  52  polymorphic positions (distributed all over the sequence) have been found i n the four H E T - C wild-type proteins. Protein variability ranged from one (between cl and c3) to 15 (between cl and c2) amino acid differences. A single amino acid change at position 133 (between cl and c3) and 153 (between c2 and mutant c2-2) can effectively change the specificity of het-c e.g. het-c 1 is incompatible with het-e2 and het-e3 while het-c3 is incompatible only with het-e2. The higher the degree of sequence variation between two alleles, the more differences they display in their specificity. For example, het-c 1 and het-c2 differ at 15 positions and they react with completely different het-d alleles. Some of the chimeric constructs obtained from different het-c alleles confer novel specificities. However, hybrid gene analysis did not reveal any limited region of H E T - C that is crucial in specificity determination. P. anserina het-c, like het-s but unlike N. crassa het-c, does not have a limited specificity region embedded within conserved domains. However, the P. anserina het-c is similar to all the above-mentioned systems, in that it contains a high ratio of nonsynonymous substitution over synonymous substitutions, suggesting a selective pressure to maintain polymorphism.  The het-e gene was also cloned by phenotypic expression - pools of cosmids containing the het-el allele (het-el from strain A ) were transformed into a het-e4 (a neutral, inactive allele) A  strain and transformants were tested in a barrage test for incompatibility with a het-c2 strain (Saupe et al., 1995a). The 1356-aa H E T - E 1 polypeptide contains a GTP-binding domain and a A  WD40-repeats region; it is the first report of these two domains being present in the same protein. The H E T - E 1 GTP-binding domain contains the conserved P-loop, G 2 , G 3 , G 4 motifs, A  suggesting that it belongs to the GTPase superfamily (Bourne et al., 1991). This GTP-binding domain is functional and is crucial in H E T - E reactivity - substitution of a conserved lysine of Ploop (K306S) involved in hydrogen-bonding with phosphates of G T P / G D P can abolish both GTP-binding activity and reactivity in het-c/e incompatibility (Saupe et al., 1995a; Espagne et al, 1997).  53  The W D 4 0 repeat is a protein-protein interaction domain first identified in the (3-subunit of transducin (a trimeric G-protein responsible for phototransduction i n animal) and is found in all GP proteins (and other proteins with various functions) (Neer et al., 1994). Contrary to other members of the W D 4 0 family, the 10 W D 4 0 repeats in H E T - E 1 show exceptionally high A  conservation (with over 70% identity compared to the usual 20-30% range). The high degree of sequence conservation may provide a means for unequal crossing over that may lead to polymorphism in the number of repeats in wild-type alleles of het-e. It was found that the number of W D 4 0 repeats varies from 3 to 12 in wild-type isolates (Saupe et al., 1995a; Espagne et al., 1997). H E T - E specificity cannot be solely dependent upon the number of repeats, since there is no apparent correlation between numbers of repeats and specificity (Saupe et al., 1995b). However, analysis of reactivity of wild-type and UV-mutant alleles demonstrated that the reactive het-e alleles contain at least ten W D 4 0 repeats (Espagne et al., 1997). Deletion of one repeat (as shown by U V mutants and in vitro deletion constructs) is sufficient to inactivate het-e incompatibility reactivity. The requirement of such a high number of W D 4 0 repeats (the normal range is 4 to 8) for reactivity suggests that H E T - E may be associated with many different proteins, which may be necessary for correct assembly and function. A s H E T - E contains functional domains found in both a and P-subunits of trimeric G proteins, it may define a new class of proteins involved in signal transduction. Disruption of het-e by gene replacement does not reveal further information about H E T - E functionality; a het-e knock-out strain behaves as a null incompatibility allele but otherwise displays wild-type phenotype (Espagne et ai., 1997). However, the lack of altered phenotype (such as colonial growth and female sterility as found in het-c mutants) in the het-e-mx\\ strain could be due to the presence of het-e-re\ated genes found in the genome (Espagne et al., 1997).  54  1.4.7 P. anserina idi genes (induced during incompatibility) L i k e the het-c/d and het-c/e systems, the P. anserina het-r and het-v loci mediate nonallelic incompatibility (Bernet, 1967b). The two loci each have two alleles (R/r and V/Vl, respectively), with the het-R het-Valleles  being the antagonistic combination. The het-R/V  system is unique in the sense that the lethality of the incompatibility reaction is conditional - it is triggered at 26°C but not at 32°C (Labarere, 1973). This feature has allowed the biochemical characterization of the incompatibility reaction. For example, the autolytic reaction of the het-R het-V self-incompatibility (SI) strain at non-permissive temperature (26°C) is correlated with a decrease in R N A production (Labarere et al., 1974), an appearance o f new proteins (Boucherie et al., 1981) and an increase in proteolytic and other enzymatic activities (Begueret & Bernet, 1973; Boucherie & Bernet, 1978). Two-dimensional electrophoresis of in virro-translated m R N A products from autolytic het-R het-V strain has identified at least 11 polypeptides preferentially expressed during induction of incompatibility (Bourges et al., 1998). Three idi genes which are induced during incompatibility were isolated using a subtractive c D N A library enhanced for m R N A s which are more abundant in the het-R het-V SI strain 8 hours after the shift to non-permissive temperature (Bourges et al., 1998).  Northern analysis verified that the expression of idi-1, idi-2 and idi-3 genes is highly induced during het-R/V incompatibility reaction. A l l three IDI polypeptides are small proteins (201, 157 and 196-aa) with putative signal peptides. The expression o f two idi genes is strictly associated with cell-lysis mediated by non-allelic (and not allelic) incompatibility. The induction  of idi-1 and idi-3 is observed in het-R het-V and het-C het-E strains but not in an het-s het-S strain. The induction is eliminated in strains that are suppressed for non-allelic incompatibility  (het-R het-V mod-Al mod-Bl and het-R het-V mod-Cl; see below). The expression of idi-1 and idi-3 is not enhanced during stress (heat shock) conditions, therefore it is likely that IDI-1 and IDI-3 are directly involved in the cell death reaction rather than merely induced as a consequence of cell death reaction. Recently, a GFP-localization study demonstrated that all three idi gene  55  products are located in the septa during incompatibility cell-lysis. Since plugging of septal pores is a hallmark feature of dying incompatible cells (Beckett et al., 1974), presumably to confine the disintegrating cells and prevent the spreading of incompatible components, the idi genes are very likely candidates for cell disintegration and/or hyphal compartmentation during vegetative incompatibility.  1.4.8 Suppressor (modifier) genes for heterokaryon incompatibility in P. anserina A s mentioned above, a cross between non-allelic incompatible strains yields occasional uninucleate progeny containing the two non-allelic antagonistic genes (e.g. het-c2 and het-el). Such progeny are inhibited upon germination and are subsequently destroyed in a generalized lytic reaction (Bernet et al., 1973). Some of these self-incompatible (SI) strains, however, can escape from restricted growth and emerge as wild-type sectors. M o s t of the escape mutations occur at het loci, but some occur at unlinked loci that give a modified self-incompatible (MSI) phenotype; the apical growth of the SI strain is restored (although with less dense mycelium and fewer aerial filaments) while the lytic reaction is only partially suppressed (Belcour & Bernet, 1969; Bernet et al., 1973). M S I strains also exhibit cold sensitivity (failure to grow at 11 °C), which can be suppressed by mutations in the mod-B locus (Bernet et al., 1973). A mutation at mod-C, which specifically suppresses both growth inhibition and lysis of incompatible het-R/V (but not other allelic or non-allelic) incompatibility, has also been isolated (Labarere & Bernet, 1977).  Modifier mutations at one locus, mod-A, give such an M S I phenotype (Belcour & Bernet, 1969). The mod-Al mutation is recessive and only suppresses growth inhibition of non-allelic (het-c/d, het-c/e and het-r/v) but not allelic incompatibility systems (Becour & Bernet, 1969; Bernet et al., 1973). Self-lysis in the mod-Al M S I strain can be suppressed by mod-Bl - mod-Al mod-B 1 double mutant in SI background has normal growth with no lytic defect (Bernet, 1971; Labarere, 1973). Although no altered phenotype is observed for single mod-Al or mod-Bl  56  mutants i n a compatible background, the mod-Al mod-Bl double mutant has a defect in protoperithecia formation and is female sterile, most noticeably in unfavorable growing conditions (such as exhaustion of nutrients) and in certain combination of het backgrounds (Bernet, 1971, 1992; Boucherie & Bernet, 1974, 1980).  The mod-A gene was cloned from a cosmid library by complementation of the mod-Al mod-Bl female sterility defect (Barreau et al., 1998). The 687-aa M O D - A is not essential; a strain with mod-A gene deleted confers a typical mod-Al phenotype ( M S I and female sterile i n mod-Bl background). M O D - A contains a proline-rich region with a potential SH3-binding domains; S H 3 domains are found in all kinds of signaling molecules such as kinases and phosphatases (Cohen et al, 1995). Many fungal proteins containing S H 3 domain or SH3 ligand motifs are involved i n morphogenesis (Barreau et al., 1998). It is possible that M O D - A is involved i n a signal transduction pathway that affects female organ formation during nutrient depletion. A model to explain the female sterility data is that for P. anserina to gather growing potential for protoperithecial formation, growth arrest (mod-A function) coupled with cell lysis (mod-B function) of hyphae surrounding the fruiting bodies are required (Barreau et al., 1998). mod-A may also affect regulation of protease activity. Protease III and I V activities, which are responsible for most o f the proteolysis i n het-R het-V cells, is absent i n m o d - A mod-B double mutant and mod-A mutation eliminate proteases C activity i n certain physiological condition (Begueret & Bernet, 1973). Expression of the pap A gene (encoding an aspartyl protease expressed during carbon starvation) is also strongly reduced i n the m o d - A l background (Paoletti et al., 1998).  Mutations at the mod-D locus have been isolated by their suppression of lytic reaction. m o d - D l suppresses the lytic phenotype of a M S I strain (het-C het-E m o d - A l m o d - C l ; Labarere & Bernet, 1979a), while mod-D2 and mod-D3 mutants were selected for their autolyticinhibition i n the self-lytic m o d - A l mod-B 11 strain (Durrens et al., 1979). Mutations at mod-D  57  cannot suppress the lytic reaction by itself, but mod-D mutants have defects in aerial hyphal growth, protoperithecial formation, spore germination and secondary ramification (Durrens et al., 1979; Labarere & Bernet, 1979a, b). mod-D was cloned by complementation of both vegetative and sexual defects i n a m o d - D l strain (Loubradou et al., 1999). The 354-aa M O D - D polypeptide is similar to the a-subunit of an heterotrimeric G protein. The less severe m o d - D l and mod-D3 mutations probably result in a decrease in c A M P levels since supplements of c A M P can restore vegetative growth in strains with these mutations. This result agrees with previous data that an extra copy of PaAC (which encodes an adenylate cyclase) can relieve the vegetative defect of mod-Dl (Loubradou et al, 1996). The involvement of mod-D in vegetative incompatibility was confirmed since either mod-Dl or mod-D2 can partially restore growth of a cold-sensitive M S I strain (het-C het-E mod-Al) at 11 °C (Loubradou et al, 1999). However, the suppressor effect of mod-Dl is probably due to its induction of defects in a signal pathway other than the c A M P pathway since increase in c A M P concentration does not attenuate the growth restoration due to mod-Dl mutation in the het-C het-E mod-Al mod-Dl strain.  The mod-El mutation was selected on its ability to restore growth renewal from the quiescent stage in the mod-D2 mutant (Durrens, 1982). It was later shown to suppress all developmental defects of the mod-D2 mutation (Durrens & Bernet, 1985). The Mod-El mutant is thermosensitive (cannot grow at 35°C), defective in glucose-dependent growth control (has excessive dry weight production and reduced life span following glucose exhaustion) and has spontaneous development of (sterile) perithecia from protoperithecia in the absence of fertilization (Durrens, 1983). The mod-E gene was cloned by its ability to complement growth of the mod-El mutant at 35°C (Loubradou et al, 1997). The 701-aa M O D - E is a member of the Hsp90 family and is responsive to heat-shock condition - mod-E expression is strongly induced at elevated temperature (37°C). Further characterization of mod-E demonstrated that it is involved in both sexual development and vegetative incompatibility - (i) a mod-El x mod-El cross contains fewer asci with mature spores and gives a dramatically higher frequency of 58  uninucleate spores (50% vs. 1-5% in a wild-type cross); (ii) mod-El mutation partly restores growth of SI het-R het-V strain at non-permissive temperature (with similar radial growth rate as wild type but with less dense mycelium and presence of lysed cells); mod-El has no effect on het-c/d or het-c/e incompatibility system. The involvement of mod-El with het-R/V system agrees with previous finding that the mod-C mutations, which suppress the incompatibility reaction in a SI het-R het-V strain (but do not affect other incompatibility systems; Labarere & Bernet, 1977), are epistatic to mod-El growth and developmental defect (Durrens & Bernet, 1985). Based on the mod-El phenotypes and the fact that other Hsp90 proteins are implicated in cell cycle control, M O D - E could be involved in negative control of cell cycle (e.g. by association with a protein kinase) that affects both sexual development and vegetative incompatibility (Loubradou et al., 1997).  59  2. Functional Analysis of mat A-l Gene of Neurospora crassa Some of the results described in this chapter are published in Saupe et al. (1996b).  2.1 INTRODUCTION Mating type in N. crassa exists as two alternative forms, A and a, which interact to initiate the sexual cycle (Dodge, 1935). The mating type locus also acts as a het locus (Beadle & Coonradt, 1944; M y l y k , 1976b); A and a hyphae cannot fuse and grow as a vigorous heterokaryon. The A and a sequences occupy the same locus but are dissimilar (Glass et al., 1988) and have been termed idiomorphs (non-homologous alleles; Metzenberg & Glass, 1990). The mat a locus contains two O R F s , mat a-l and mat a-2 (Staben & Yanofsky, 1990; Pbggeler & Kiick, 2000). The mat a-l gene provides all functions for the a mating type (mating, postfertilization and heterokaryon incompatibility) (Staben & Yanofsky, 1990; Chang & Staben, 1994). The function of mat a-2 is unknown - it is non-essential to mating and sexual development and could represent a non-translated pseudogene (Poggeler & Kiick, 2000). The mat A locus of N. crassa contains three O R F s : mat A-l, A-2 and A-3 (Glass et al., 1990; Ferreira et al., 1996). M A T A - 2 and M A T A - 3 are responsible for post-fertilization events (ascospore formation) (Ferreira et al., 1998). The mat A-l gene is both necessary and sufficient to confer mating specificity of A and trigger heterokaryon incompatibility with a (Glass et al, 1990). Mutations i n an unlinked tol locus suppress mating-type incompatibility such that A tol and a tol strains form a vigorous heterokaryon (Newmeyer, 1970). The mating-type-associated incompatibility is therefore mediated by the gene products o f mat A-l, mat a-l and toV'.  The 293-aa M A T A - l contains a region of similarity (oc-domain) with the M A T a l mating-type protein of S. cerevisiae (Tatchell et al, 1981). The a-domain is also found in other mating-type polypeptides, such as M A T - 1 (of C. heterostrophus; Turgeon etal, 1993) and F M R - 1 (of P. anserina; Debuchy & Coppin, 1992) (see section 1.3). M A T a l is a positive transcriptional factor that activates the expression of a specific genes (in cooperation with 60  M C M 1 ; Passmore et al, 1988), such as MFal/MFa2  (encode a-factor precursor; Kurjan &  Herskowitz, 1982) and STE3 (encodes a-factor receptor; Sprague et al., 1983). In order to delineate the functional domains of mat A-l, a series of nonsense and frameshift constructs were tested for their male-mating activity and heterokaryon incompatibility.  2.2 MATERIALS AND  METHODS  2.2.1 Strains, media and culturing Culturing and crossing, using V o g e l ' s (Vogel, 1964) and Westergaard's (Westergaard & Mitchell, 1947) media, respectively, were performed as previously described (Davis and Deserres, 1970; Perkins, 1986). Wild-type Oak Ridge A strain (Fungal Genetics Stock Center F G S C 2489) and previously obtained A sterile mutants were used for D N A isolation; un-3 adm  3A nic-2 cyh-1 A  m42  cyh-1 A  m54  ( F G S C 4569), un-3 ad-3A nic-2 cyh-1 A  ( F G S C 4571), un-3 ad-3A nic-2 cyh-1 A  m44  m56  ( F G S C 4570), un-3 ad-3A nic-2  ( F G S C 4572) and un-3 ad-3A A  m64  (FGSC  4573) originated from Griffiths (1982). The un-3 ad-3A nic-2 cyh-1 A " strain was isolated by m  Saupe et al. (1996b) according to Griffiths (1982). The homothallic Neurospora strain N. pannonica ( F G S C 7221) was also used, fl a ( F G S C 4347) and/7 A ( F G S C 4317) were used for mating assays (Perkins etal., 1989). C15-1 (pan-2; inl; arg-5 a; M . L . Smith), C15-2 (pan-2; arg-5A;  M . L . Smith), 1-10-1 (ad-3A nic-2; tol a; A . J . F . Griffiths) and 1-20-41 (ad-3B arg-1; tol  A; A . J . F . Griffiths) were used for spheroplast preparation and transformation experiments.  2.2.2 DNA isolation and PCR amplification Genomic D N A isolation was performed according to Stevens & Metzenberg (1982). P C R amplification (Saiki et al., 1988) from genomic D N A was performed using primers that span mat A-l (base 3802-3826 and 4843-4867) (Glass et al, 1990) in a model 480 Perkin-Elmer D N A cycler. P C R products were cloned into the P C R I I vector using the T A cloning kit (Invitrogen, San Diego, C A ) .  61  2.2.3 Plasmid construction for mat A-l and FMR1 PCR-amplified mat A-l mutant alleles (as a 1.1 kb BamHI fragment) were subcloned from the P C R I I vector into p O K E 3 vector carrying the pan-2 marker (Grotelueschen & Metzenberg, unpublished results) and into the pCB1004 vector carrying the hygromycin-resistant phosphotransferase (hph) gene (Carroll et al, 1994). Escherichia coli D H 5 a (Hanahan, 1983) was used as bacterial host strain for plasmid amplification.  T o construct a Neurospora-compatible  plasmid containing the P. anserina FMR1 gene, a  2.3 kb KpnUXhol FMR1 fragment (from plasmid p B P L P - 0 ; Debuchy et al, 1993) was cloned into pCB1004 vector. The final construct is named F M R l / p C B . The F M R l ( A 1 5 5 - 3 0 5 ) / p C B plasmid is constructed by deleting a 3' EcoRV fragment from the F M R l / p C B plasmid.  In order to build a pCB1004 plasmid containing an over-expressing FMR1 gene, a 3.5 kb EcoRVXbal  fragment of FMR1 gene (from plasmid P p D " F M R l / p U C 1 8 ; gift from R . Debuchy) G  fused with a strong promoter from A. nidulans glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al, 1988) was cloned into pCB1004. The construct is named P p D " F M R l / p C B . G  2.2.4 Site-directed mutagenesis and chimeric construction T o introduce the L 1 0 9 T mutation in mat A-l, a 1.1 kb BamHI mat A-l fragment was cloned from the mat A - i / p C B vector into the smaller p U C 1 9 plasmid and was subjected to sitedirected mutagenesis using the U . S . E . mutagenesis kit (Pharmacia, Bale d'Urfe, P Q ) following the manufacturer's protocol; oligo-based mutagenesis has poor efficiency when performed in a large plasmid. Primer L R R 5' T G A A T C C A G G T T T G C A G A G T , which contains a novel BstNl restriction site for screening, was used to introduce the L 1 0 9 T mutation. The Seal selection primer 5' C T G T G A C T G G T G A C G C G T C A A C C A A G T C 3' (Pharmacia, Bale d'Urfe, PQ), which converts a Seal site to Mlul site in the amp gene carried in the p U C 1 9 plasmid, was used to enhance selection of mutant plasmid. After the mat A - 7 / p U C 1 9 single-stranded D N A was  62  annealed to both primers and had the second strand synthesized, the plasmids were cut by Seal enzyme such that non-mutated plasmids were linearized and eliminated. The mutant plasmid was recovered after one round o f selection in N M 5 2 2 mutS E. coli cells and the site-directed mat A-l mutation was verified by D N A sequencing (Sanger et al, 1977). The mutant allele was cloned into p O K E 3 vector and was subjected to transformation assays.  Chimeric constructs were made between mat A-l and FMR1. However, since there is no convenient restriction site between the D N A sequences that encode the a-domain and the first L R R (i.e. between amino acids V92 and Y93), an artificial Bst 11071 site was introduced into both mat A-l and EMR1 in order to facilitate construction. Primer N C 9 2 5 ' C A T G T G C T C G G T A T A C T C G T C A A T C C G G 3' and primer P A 9 2 5' C A T G T G T G C A G T A T A C T C T G C G A T T C G C 3' were used to introduce a novel fisf 11071 restriction site in the plasmids mat A - 7 / p U C 1 9 and FMR1VpUC19,  respectively. Site-directed mutagenesis was performed as described above.  Mutated plasmids were recovered after N M 5 2 2 mutS selection and their 3' Bstl IQll-Xbal fragments were swapped. T w o plasmids containing reciprocal chimeric constructs, the U F A 9 2 (pUC19 vector containing aa 1-92 of F M R 1 and aa 93-end of M A T A - l ) and U A F 9 2 (pUC19 vector containing aa 1-92 of M A T A - l and aa 93-end of F M R 1 ) , were obtained. The chimeric genes were cloned into hygromycin-resistant p C B 1 0 0 4 and the plasmids were named C F A 9 2 and C A F 9 2 , respectively.  2.2.5 D N A sequence analyses D N A sequences were determined using the A B I automated Taq DyeDeoxy Terminator cycle method (Mississauga, O N ) at the N A P S unit (Biotechnology Laboratory, University o f British Columbia). Computer sequence analyses for protein and D N A were done using the G C G package available from the Wisconsin Genetics Computer Group (Devereux et al., 1984).  63  2.2.6 Transformation reduction assay for heterokaryon incompatibility Competent spheroplast preparation and transformation were performed as previously described (Schweizer et al, 1981; Vollmer & Yanofsky, 1986). The transformation reduction assay for heterokaryon incompatibility was done according to Glass et al. (1988). To test if the mat A-l mutants contain the incompatibility activity, the pOKE3 constructs were introduced to a (C15-1) and A (C15-2) spheroplasts. To test if tol suppresses transformation reduction activity, the pCB1004 constructs were introduced into the tol a (1-10-1) and tol A (1-40-1) spheroplasts.  2.3 RESULTS 2.3.1 PCR amplification and construction of various mat A-l (A ) mutant constructs m  To delineate the functional domains of mat A-l, a series of mutant mat A-l alleles were tested for their ability to confer mating and incompatibility function. Genomic DNA from wild type and various frameshift and nonsense sterile mutants of mat A-l (A , m54  and A ) m44  A , m64  A , m42  A ", m  (Griffiths, 1982; Saupe etal, 1996b) was isolated and subsequently PCR-amplified  using two primers that bracket mat A-l (bases 3802-3826 and 4843-4867; Glass et al, 1990). The wild type mat A-l allele encodes a polypeptide of 293-amino acids while the mutant alleles encode the first 163 (A™ ), 111 (A ), 99 (A ), 85 (A ) and 52 (A ) amino acids of MAT A - l 54  m64  m42  m99  m44  protein, respectively. The 1.1-kb PCR products of mat A-l were obtained for each strain and were subsequently cloned into the PCRII vector. To ensure that the PCR reactions amplified the mat A-l gene, the PCRII vectors containing mat A-l clones were tested for the presence of mat A-l gene using Southern blot analysis (Southern, 1975) with the entire A idiomorph (Glass et al, 1988) clone as a probe. Southern blot results showed that mat A-l was successfully amplified (data not shown).  To obtain a Afewraspora-compatible vector containing the various constructs, the 1.1 kb BamHl mat A-l fragments from wild-type and each of the five mutant alleles were subcloned  64  from the P C R I I vector into the p O K E 3 vector carrying the pan-2 marker and into the pCB1004 vector carrying the hygromycin resistance gene.  2.3.2 Incompatibility of mutant mat A-l alleles in a transformation assay To test i f the mat A-l mutant alleles retain heterokaryon incompatibility activity, the subclones (in p O K E vector) were introduced into a spheroplasts. If a mat A-l mutant allele confers heterokaryon incompatibility activity, then the transformation frequency should be significantly reduced, because transformants containing functional mat a-l, mat A-l and tot do not regenerate (Glass et al, 1988). To demonstrate that the transformation reduction is due to mating-type incompatibility and that tol suppresses incompatibility activity in these mutant alleles, the pCB1004 constructs were introduced into the tol a and tol A spheroplasts. The P C R cloning and transformation strategy is summarized in Figure 2-1.  The results of the transformation experiment are shown i n F i g . 2-2 and summarized in Figure 2-3. Three of the constructs, A , m42  A " and A , m  transformed the a and A recipient strains  with similar efficiencies. However, when the A  m44  m64  and A  m54  constructs were introduced into the a  recipient strain, a significantly reduced transformation frequency was obtained. In three separate replicates, an average of 30 transformants (per 10-cm petri dish) were recovered following the transformation of the A  m64  and A  m54  alleles into the a spheroplasts, as compared to an average of  300 transformants recovered when A spheroplasts were recipients. In contrast, an average of two transformants are recovered when the wild-type mat A-l allele was introduced into the a spheroplasts.  65  matA-  mat A-3  - matA-i— Genomic DNA PCR  PCRII vector  Clone  A-1  PCB1004 vector HygR Transform to Hyg",  Transform to Pan cell (A and a)  tol' cell (A and a)  A cells: normal  a cells: normal  A cells: normal  a cells: transformation reduction  Figure 2-1. Strategy for transformation reduction assay for heterokaryon incompatibility. Wild type and various A alleles of the mat A-l gene were amplified by PCR and were later tested for their ability to trigger mating-type associated incompatibility. If a mat A-l construct contains incompatibility function, its introduction into a tol recipient will lead to dramatic decrease in number of transformants as compared to transformation into A tol background. On the other hand, the transformation reduction should not be observed in a tol' background since the tol mutation suppresses mating-type-associated incompatibility. m  +  +  Figure 2-2. Results of the introduction of mutant mat A-l alleles (A ) into a and A recipients m  strains. Results correspond to the mean of three independent experiments. The transformation frequency for each transformation test is illustrated as a percentage of the transformation efficiency o f vector (no insert) control. Transformation frequency of a hundred percents correspond to the number of transformants can be obtained when the vector control gives 100 transformants per 200 ng of D N A (introduced into 4 x 10 spheroplasts) i n a transformation test. 6  For the vector control, the typical number of transformants per plate (500 ng of D N A into 10  7  spheroplasts) is roughly 250.  67  INC  52  72  m99  A  85 *m42  m64  &  Am54  163 187  matA-1  293  Figure 2-3. Mutant and truncated matA-1 alleles: topography of presumptive translational products and their properties. The position of the A mutations in the mat A-l ORF is indicated m  by arrowheads together with the nature of the mutation, frame shift (-2, +1, etc) or stop codon (*). The position of the last amino acid corresponding to the original matA-1 sequence is given. The solid area represents the regions of the open reading frame translated out of frame before the novel stop codon. On the right, the results heterokaryon incompatibility (INC) assays are given. Incompatibility was assayed by introducing cloned mutant alleles into an a recipient. A construct is determined to confer mat A-l heterokaryon incompatibility function (+) if its introduction into a spheroplasts lead to a reduction in transformation efficiency.  68  Based on the results of the transformation assay, an ectopic matA-1 containing only the first 111 amino acids is sufficient to elicit the mating-type incompatibility function when the A and a sequences reside in the same nucleus. DI, D2, D3 and D4, four in vitro deletion constructs truncated at amino acid positions 288, 227, 184, and 163 respectively, also confer incompatibility functions as tested by transformation assay (Saupe et al., 1996b). These data are in agreement with the A mutant phenotypes such that mat A-l constructs containing at least the m  first 111 amino acids retain incompatibility function.  The tol mutation suppresses the mating-type incompatibility in both partial diploids for mating-type and mixed mating-type heterokaryons (Newmeyer, 1970). The A mutant alleles m  and the wild-type mat A-l were introduced into tol a and tol A strains to determine if the tol mutation can suppress the incompatibility as assayed by transformation experiments. Transformation efficiencies into tol a and tol A recipients were comparable for all A mutant m  alleles and the wild-type matA-1 (averaging 200-300 transformants per plate), demonstrating that the reduction in transformation frequency as observed for the case of matA-1, A  m64  and A  m54  was due to triggering of mating-type-associated incompatibility.  By examining the amino acids sequence flanking 100 to 111, a putative leucine-rich repeat (LRR) was identified (a second one was found further downstream; Figure 2-4A). LRRs, which have been found in numerous proteins — nuclear, cytoplasmic, and secreted, and are thought to mediate protein-protein interactions (Kobe & Deisenhofer, 1994). Each LRR contributes an exposed P-sheet that could participate in interactions with other proteins. Two LRRs are also found in MAT a-1 (Figure 2-4B). A 5-amino acid deletion within the first LRR of mat a-1 led to the loss of heterokaryon incompatibility without affecting mating activity and DNA binding in vitro (Philley & Staben, 1994). If the sequences depicted in Figure 2-4C are functional LRRs, then they may represent important protein-protein interaction domains required for the heterokaryon incompatibility reaction. 69  A.  INC 100  CYR1 LRR N. N.  crassa terricola  N.  africana  N.  pannonica  P.  anserina  150  110  PXXaXX LXXLXX LXaXXXX aXXa YSSIRTYLEQEKVTLQLWIHYAVGHL YSSIRTYLEQEKVTLQLWIHYAVGHL YSSIRTYLEQEKVTLQLWIHYRVRHL YSAIRTYLEEEKVNLQLWNHYSVGHL YSAIRDQLAEQNVTLQTWIQFAVTPL  B.  160  170  PXXaXX LXXLXXLXaXXXXaXX a PL VQHNLQPMNGLCLLTKLLESGL PL VQHNLQPMNGLCLLTRCLESGL PL VQHNLQPMNGLCLFTKCLESGL PL VQHNPPPMDGLCLLTKLLESGL PD IRYHLQPMNGLGLFLSCLNGGL  INC 200 I  CYR1 LRR  370 I  210 I  380 I  PXXaXX LXX LXXLXaXXXXaXX a RRRVSPYLKIKLLNYDVNGNLLWGTV  PXXaXXLXXLXXLXaXXXXaXX a PLRLLSLR LW LMIPLTQLSFPL  MAT A - l  93 150 MAT a - 1 194 361  YSSIRT LEQEKVTLQLWIHYAV GHL PL VQHNLQPMNG LCLLTKL LESGL RRRVSPYLKIKLLNYDVNGNL LWGTV PLRLLS LR LW LMIPLTQ LSFPL  118 173 219 382  Consensus  P--a  MAT  a-1  L  L--L-a-a  a  a  Figure 2-4. Leucine-rich repeats in mating-type proteins. A . Leucine-rich repeats (LRRs) found in Neurospora crassa M A T A - l and its homologs in other fungal species. Numbers indicate the amino acid position according to N. crassa M A T A - l protein. The top sequence (CYR1 LRR) depicted the consensus sequence found in yeast adenylate cyclase, where "a" represents V, L or I and " X " represents any amino acid (Kataoka et al, 1985). Podospora anserina FMR1 is the only protein listed that does not confer heterokaryon incompatibility function. Position 100 to 111 is required for incompatibility (INC) function in M A T A - l . The a-domain of M A T A - l is found at position 42 to 89 (not shown), 4 amino acids upstream of the first L R R . B. LRRs identified in Neurospora crassa M A T a-1. Deletion of amino acids from 216 to 220 or a R258S substitution (not shown) in M A T a-1 abolishes heterokaryon incompatibility (INC) but does not affect mating nor D N A binding in vitro (Philley & Staben, 1994). The H M G box is found at position 115 to 185 (not shown), 9 amino acids upstream of the first L R R . C . Leucine-rich repeats (LRR) within the mating-type protein of N. crassa are implicated in the heterokaryon incompatibility function. Two LRRs from each mating-type gene and their consensus (bottom line) are depicted. Deletion or change of the underlined residues abolishes incompatibility but does not affect mating function.  70  2.3.3 Construction of chimerics between mat A-l and FMR1 The P. anserina FMR1 (fertilization minus regulator) gene confers mating identity for the mat- strains and is a homolog of N. crassa mat A-l (Debuchy & Coppin, 1992) (see section 1.3.4). The amino terminal portions of the two proteins are very similar (Figure 2-5). The two mating-type proteins are functional homologs since the introduction of P. anserina FMR1 confers mating in N. crassa and the introduction of N. crassa mat A-l confers mating in P. anserina (Arnaise et al., 1993). In P. anserina, a pseudohomothallic ascomycete, a germinating spore contains both opposite mating type nuclei (mat+ and mat-). The two nuclear types grow as a heterokaryon and therefore do not exhibit mating-type-associated incompatibility, although incompatibility mediated by other het loci is observed P. anserina.  In order to test if the FMR1 gene confers heterokaryon incompatibility in N. crassa in a transformation reduction assay, the FMR1 gene was cloned into a Atewros/wra-compatible plasmid, pCB1004 (containing a hygromycin resistance gene) (Carroll et al., 1994). The F M R l / p C B construct was then subjected to the transformation assay as described above. Transformation results showed that the F M R l / p C B plasmid has comparable transformation frequencies when introduced into A and a spheroplasts (Figure 2-6). These data confirmed that the FMR1 gene does not confer heterokaryon incompatibility (with mat a-l) in N. crassa, although it confers mating with N. crassa a strain. To ensure that the failure of FMR1 to confer heterokaryon incompatibility is due to the alteration in amino acid sequence and not because of the low expression of FMR1 in N. crassa, or the masking of the incompatibility domain by carboxyl terminus (which is different than that of M A T A - l ; see Figure 2-5) through steric hindrance, two additional FMR1 constructs were tested in the transformation reduction assay. The first construct is made from a gene fusion of FMR1 with a strong promoter from the A. nidulans glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al, 1988). The second construct was made by deleting a 3' fragment from the FMR1 construct so that the carboxyl  71  10 20 yEGVIXffVKimDLAEDDEEr^  N.  crassa  P.  anserina  30  40  ly&GINSILQIFEGKSGDRAEn?IKVLSEMMREGIER  60  70  80  50  QBiKKKWGF  90  100  140  150  MSF^SYYSPLFSQI^ERSPFMITmQII^FHN^  110  120  130  K)EKyiLOIJAni3Y£^^ JgOSIVILOTmOFAVIPLCTArrR^  160 170 IVOITOOPMNXCII^^  180  190  200  IIRYHLQPMECLGIFIi? 210 RQQG  220 230 240 250 Hfi^JTDESEVGVSSMF—PRNHIVAAEWDGIINL P— L  KE PGTFDTM3GF RG1AKQNPAL AMSSL FQLP DIDPLIAQ3VGMYEFHSW 260 SHWIQ23EFGIES  270  280 290 GYSAQFEItLDSIL  300 EN3i  32 P\O^GMPFTTVPPMESHSHFIlW 310 ASSNDPYNM 3DPNKPQGF  Figure 2-5. Mating-type amino acid comparison between Neurospora crassa M A T A - l and Podospora anserina F M R 1 (Glass et al, 1990; Debuchy & Coppin, 1992). The leucine-rich repeats ( L R R s ) within the two proteins are underlined (positions 93-118 and positions 150-173). The a-domain is found i n position 42 to 89 and is italicized.  "|" indicates an identical amino  acid. ":" indicates a conservative change. "." indicates a semi-conservative change.  72  terminal portion o f the protein (from H455 to the end) was eliminated. Transformation reduction assays showed that neither o f the F M R l ( A 1 5 5 - 3 0 5 ) / p C B nor the P G P D : : F M R l / p C B constructs conferred transformation reduction when introduced into N . crassa a spheroplasts.  These data  agreed with previous results by Arnaise et al. (1993), which have shown that when FMR1 (and FPR1) were introduced into N crassa, incompatibility reaction was not elicited.  Since FMR1 confers mating activity in Neurospora as mat A but does not confer heterokaryon incompatibility, it can be viewed as a mat A-l mutant in the heterokaryon incompatibility function. However, as shown in Figure 2-5, although matA-1 and FMR1 are similar, there are many amino-acids differences between the two polypeptides. It was impossible to identify differences between the two polypeptides that can correlate the presence of het function in matA-1 but the lack of it in FMR1. Chimeric proteins between M A T A - l and F M R 1 were therefore the next logical approach to pinpoint the heterokaryon incompatibility domain.  From the transformation test of the A strains, a L R R - l i k e sequence in M A T A - l is m  absolutely required for mediating heterokaryon incompatibility. T o examine i f the L R R sequence is functionally important in mediating incompatibility (and not merely enhancing the folding of a-domain or increasing m R N A stability i n the A  m64  and A  M54  mutants), chimerics  between M A T A - l and F M R 1 with a fusion point at the beginning of the first L R R (i.e. between amino acids V92 and Y93) were constructed. Since there is no convenient restriction site, an artificial BstllOll  site was introduced into both matA-1 and FMR1 clones in order to facilitate  chimeric construction. T w o plasmids containing reciprocal chimeric genes, the C F A 9 2 (pCB1004 vector containing aa 1-92 of F M R 1 and aa 93-end of M A T A - l ) and C A F 9 2 (pCB1004 vector containing aa 1-92 of M A T A - l and aa 93-end of F M R 1 ) , were constructed and used in the transformation reduction assay.  When introduced into N. crassa competent cells, the C A F 9 2 plasmid exhibited similar transformation efficiencies in A and a spheroplasts, wheras the C F A 9 2 plasmid exhibited 73  considerably lower transformation frequency (20 fold) in the a recipient (Figure 2-6). These data indicated that the C F A 9 2 chimeric (containing the M A T A - l L R R ) confers heterokaryon incompatibility in N. crassa whereas C A F 9 2 (containing the F M R 1 L R R ) does not. Furthermore, when C F A 9 2 and C A F 9 2 were introduced into a tol a recipient (1-10-1), C F A 9 2 and C A F 9 2 - transformants conferring dual mating activity can be recovered (i.e. they initiated peritherial development with A and a tester strains). These data demonstrated that functions of mating and incompatibility in M A T A - l can be separated and that the absence of incompatibility function in C A F 9 2 is due to lack of an incompatibility domain and not due to global mis-folding of the protein. The position of the breakpoint in the two chimeric constructs and their transformation/mating results shows that the difference in the L R R sequence could be responsible for the lack of incompatibility function in FMR1 gene.  2.3.4 Site-directed mutagenesis of LRR in matA-1 Results from the transformation assay of chimeric mat A-1IFMR1 genes show that the difference in the L R R sequence between M A T A - l and F M R 1 could be responsible for the lack of the incompatibility function in F M R 1 . One potential candidate found in the first L R R that may be responsible for the functional difference is the L 1 0 9 T (Figure 2-4A). L109 is a conserved leucine among different Neurospora M A T A - l homologs in the first L R R . This leucine residue at position 109 is replaced by a threonine in the F M R 1 polypeptide.  To test i f the L 1 0 9 T is responsible for the loss of heterokaryon incompatibility in F M R 1 , the same amino acid substitution was introduced to the wild type mat A-l gene. The mat A-l gene was cloned into p U C 1 9 and was subjected to site-directed mutagenesis. The mutated mat A-l clone was sequenced by the dideoxy chain-termination method (Sanger et al, 1977) and was subject to transformation assay as described above. Results of the transformation assay indicated that the site-directed mutant matA-1 L109T has the same degree of transformation reduction as wild type (averaging 4 transformants in a recipient versus 200+ transformants in A recipient)  74  (Figure 4-6). Therefore, the L 1 0 9 T substitution is not solely responsible for the absence of heterokaryon incompatibility function in F M R 1 .  2.3.5 Heterokaryon incompatibility function of other mating-type protein The L 1 0 9 T site-directed mutagenesis did not reveal a disruption of incompatibility function in M A T A - l . However, there are other candidates within the L R R that could explain the "compatible" phenotype of F M R 1 . A s shown in Figure 2-4A, there are many other changes between F M R 1 and the Neurospora M A T A - l consensus within the L R R , namely S95A, T98D, Y 9 9 Q , E 1 0 1 A , Q102E, E103Q, K 1 0 4 N , H112Q, Y 1 1 3 F , G116T and H117P. One of the residues mentioned here is of special interest - S 9 5 is the only potential protein kinase phosphorylation site ( S / T - X - R / K ) in the M A T A - l protein. It is possible that the S 9 5 A change in F M R 1 leads to the absence of phosphorylation at that position and therefore no longer allows F M R 1 to mediate incompatibility.  To test i f the S 9 5 A substitution is responsible for the loss of incompatibility function in F M R 1 is possible even without the use of site-directed mutagenesis, since another Neurospora species, N. pannonica (a homothallic species; K r u g & Khan, 1991), contains the same S 9 5 A substitution (see Figure 2-4A). The N. pannonica matA-1 gene was amplified, cloned and subject to transformation assay as described in sections 2.3.1 and 2.3.2. Transformation results indicated that the N. pannonica mat A-l gene confers heterokaryon incompatibility in N. crassa (transformation reduction is as dramatic as that of N. crassa matA-1) (Figure 2-6). Therefore the S 9 5 A substitution is not solely responsible for the loss of incompatibility function in the F M R 1 polypeptide.  75  400  •j 300 C5  a A vector  a mat  A A-1  a A FMR1  a A CAF92  a A CFA92  a A L109T  a A NPA-1  Figure 2-6. Results of the introduction of various constructs into a and A recipient strains. CAF92 and CFA92 are chimerics constructed with N. crassa matA-1 and P. anserina FMR1 gene. CAF92 encodes amino acids 1-92 of M A T A - l and 93-end of F M R 1 . CFA92 encodes amino acids 1-92 of FMR1 and 93-end of M A T A - l (including first L R R of M A T A - l ) . L109T represents a site-directed mutant of matA-1. NPA-1 represents the matA-1 gene from N. pannonica.  The transformation frequency for each transformation test is illustrated as a  percentage of the transformation efficiency of the vector (no insert) control. Transformation frequency of a hundred percents correspond to the number of transformants can be obtained when the vector control gives 100 transformants per 200 ng of D N A (introduced into 4 x 10  6  spheroplasts) in a transformation test. For the vector control, the typical number of transformants per plate (500 ng of D N A into 10 spheroplasts) is roughly 250. 7  76  2.4 DISCUSSION In heterothallic and some pseudohomothallic species, fusion of opposite mating-type reproductive structures initiates sexual reproduction. In Neurospora crassa, the mating type locus is unique in that it controls both sexual and heterokaryon compatibility (Beadle and Coonradt, 1944). Hence, although the two mating types, A and a, must fuse to go through the sexual cycle, they cannot fuse somatically and grow as a vigorous heterokaryon.  Genetic studies have provided some insights on this unique dual function. Newmeyer (1970) showed that the unlinked mutation, tol (tolerance), suppresses the mating type-associated heterokaryon incompatibility function but does not affect sexual function, thus pointing to a distinct delineation of these two roles of mating type at the cellular level. Griffiths and Delange (1978) and Griffiths (1982) obtained mutations of both A and a alleles (A and a ) by selecting m  m  for heterokaryon formation in mixtures of cells of both mating types. Most mutants were found to have lost both sexual and incompatibility functions, but one mutant, a , had lost the m33  incompatibility function and retained full fertility as a protoperithecial (maternal) and a conidial (paternal) parent. These data showed that, for the a allele, the two mating-type functions are structurally separable.  The A and a sterile mutants produce protoperithecia with trichogynes, showing that the m  m  mating-type genes do not code for structural components of the female reproductive structures. However, in a mating-type pheromone bioassay (Bistis 1981, 1983), the A and a mating-type m  m  mutants do not produce pheromone, nor do they respond to the pheromone of the opposite mating type (G. Bistis, personal communication). In S. cerevisiae, the mating identity of cells is determined by a pheromone and receptor system; a cell of a particular mating-type expresses only one pheromone and a receptor for the pheromone produced by the opposite mating-type (Banuett, 1998) (see Chapter 1.3). Binding of the pheromone to its cognate receptor facilitates  77  mating by inducing a signal transduction event that leads to the transient arrest of the cell cycle and the induction of conjugation-specific genes. It is likely that the mating type proteins in N. crassa also control expression of a pheromone/receptor system such that cells with alternate mating-type alleles can recognize each other as compatible mating partner. In support of this hypothesis, the pheromone precursor genes found in N. crassa are present in all cell types but are expressed in a mating-type specific manner (D. Ebbole and D. Bell-Pedersen, personal communication).  The matA-1 gene in A cells and mat a-1 gene in a cells are responsible for both mating and heterokaryon incompatibility; all previous A and a mutations are found in these two ORFs. m  m  Two additional genes found in the A locus, mat A-2 and mat A-3, are required for postfertilization events only (Ferreira et al., 1996, 1998). The purpose of the present study was to delineate functional regions in the matA-1 encoded polypeptide. Figure 2-7 summarizes the analyses of incompatibility function in various mutant and chimeric mat A-l constructs.  Several mutant matA-1 (A ) alleles (Griffiths, 1982; Saupe et al, 1996b) were tested for m  their ability to trigger heterokaryon incompatibility in a transformation assay. All the A  m  mutants, as generated by UV and HAP (6-N-hydroxylaminopurine) treatment, were selected for the ability to form heterokaryons with an a reference strain. All but one of the initial A mutants m  (Griffiths, 1982) were found to have frameshift mutations and have lost both male and female fertility. The exception is the A " mutant (with a nonsense mutation after aa position 85), which m  is completely male sterile but showed partial female fertility (Saupe et al, 1996b). When mated as female, A " produces 20-50 fertile, beaked perithecia on a 10-cm petri dish (as compared to m  hundreds of perithecia found in wild-type), has slower development of mature asci with ascospores (takes 15 days for asci development compared to 7 days for wild-type) and smaller number of mature asci per perithecium. The A " mutant contains all of the cc-domain, which is a m  region of homology found among various mating-type proteins, including M A T a l of S. 78  a MAT A-1  I 42  JJ 89 93 118  I  AM54 AM64  /////////:  zi  INC V  MALE FEMALE V V  293 a a V  i—n  V  X  N.D.  U  A  X  X  JJ  A  X  X  JJ  D3  LRR I 150 173  JZZE  I  D2  FMR1/A1  LRR  i—r  N.D.  V  N.D.  AM42  I  X  X  X  AM99  T  X  X  V*  X  X  X  X  V  N.D.  Am44 I  A1/FMR1  FMR1  Y////////////////////////////7-A  X  N.D.  Figure 2-7. mat A-l constructs and their functional analyses. Assayed functions include matingtype incompatibility (INC), male mating (MALE) and female mating ( F E M A L E ) activity (N.D. - not determined). Detailed topography of mutations and translational products of A mutants m  are shown in Figure 2-3. D2 and D3 are deletion constructs truncated at positions 227 and 184, respectively. FMR1/A1 and A1/FMR1 are chimeric proteins constructed from M A T A - l and FMR1. * A " mutant confers partial female mating activity - it forms few perithecia and its asci m  and ascospore development is delayed. V = presence of activity. X = absence of activity.  79  cerevisiae (Tatchell et al., 1981) (see section 1.3). MATcc-1, in cooperation with the MCM1 protein, is a transcription activator that recognize the promoter region of many a-specific genes (Grayhack, 1992). A short region within the a-domain (which has the greatest similarity among fungal mating-type proteins) is proposed to interact with MCM1 (Hagen et al., 1993; Yuan et al., 1993). Even though the region of amino acids 1-85 is intact in A , m54  A  m64  and A , m42  these mutants  are female sterile (Figure 2-7). It is possible that their out-of-frame C-terminal tails affect female fertility through steric hindrance or causes these fusion products to be rapidly targeted to proteolytic degradation (although heterokaryon incompatibility function is intact in these mutants). Alternatively, the premature stop codon in A " may be ignored during translation in m  the developing perithecia such that a small amount of full length MAT A - l is produced.  Results from the transformation of A constructs suggest that position 100-111 in MAT m  A-l is important for heterokaryon incompatibility, since the A"  and A  154  transformation reduction in an a recipient while A , A " and A  m64  m42  m  m44  alleles confer  do not (Figure 2-3). The  results from transformation of A mutants agree with the phenotype of in vitro deletion m  constructs of mat A-l, namely DI (truncated at position 288), D2 (227-aa), D3 (184-aa) and D4 (163-aa), which confer incompatibility function as tested by transformation assay (Saupe et al, 1996b). However, since all the A mutants were selected for their ability to form a stable m  heterokaryon with an a strain (Griffiths, 1982), the A  m54  and A  m64  strains have conflicting  phenotypes of incompatibility function. The same mutant matA-1 allele has lost the incompatibility function in forced heterokaryon but is still capable of triggering the incompatibility in a transformation assay. Different hypotheses can be proposed to explain this difference. Heterokaryon compatibility of the A  m54  and A  m64  mutants could be a consequence of  the instability of the chimeric MAT A - l polypeptide or of the mRNA bearing premature stop codons. Nonsense codon-mediated mRNA degradation has been reported in yeast (Peltz et ah, 1993, 1994; Hagan et ah, 1995). On the other hand, integration of transforming DNA often occurs in transcriptionally active regions of the genome and in multiple copies. This difference 80  could result in a higher expression level of the mutated alleles as transgenes, thus compensating for the instability of A  m64  and A™ mRNA. Moreover, in heterokaryons, mat A-l and mat a-l are 54  expressed from distinct nuclei whereas in transformants the transgenic mat A-l allele is expressed from the same nucleus as the resident mat a-l. Considering that mat a-l and mat A-l are putative transcriptional regulators, it is possible that in mixed mating type heterokaryons these products are preferentially targeted back to the nucleus they originated from, thus limiting their interaction when expressed from different nuclei.  The transformation assay suggests that the incompatibility function is mildly affected in A  mS4  and A .  from A  m64  m54  The reduction of transformation efficiency is 10 to 12 fold for mat A-l alleles  and A  m64  whereas the reduction is at least 50 fold for the wild type mat A-l allele.  Thus, the amino acids from 164 to 293 (including the second LRR) appear to contain sequences enhancing the incompatibility response although they do not appear to be essential. Alternatively, this region might not be involved in the incompatibility function at all; the transformation frequencies observed in experiments with A™ and A 54  mM  constructs could be  explained by mRNA instability as described above.  Amino acids from position 100 to 111, which are required for heterokaryon incompatibility, are within a putative leucine-rich repeat (LRR) (Figure 2-4A). LRRs are thought to mediate protein-protein interactions (Kobe & Deisenhofer, 1994). A second LRR is found at positions 150-173 in MAT A - l while two putative LRRs are found in MAT a-l (Figure 2-4B, C). A 5-amino acid deletion within the first LRR of MAT a-l led to the loss of heterokaryon incompatibility without affecting mating activity and DNA binding in vitro (Philley & Staben, 1994). These data demonstrated that these putative LRRs may be functionally important for heterokaryon incompatibility.  81  To investigate the importance of the LRR in mediating mating-type-associated incompatibility, chimeric constructs with a fusion point at the start of the first LRR were made from the mat A-l and FMR1 genes. FMR1 specifies mating identity for mat- nuclei in P. anserina and has strong homology with MAT A - l (Debuchy & Coppin, 1992). The two matingtype proteins are true functional homologs since they confer mating activity as heterologous transgenes (Arnaise et ai, 1993). FMR1, however, does not confer incompatibility in N. crassa as assayed by transformation (see section 2.3.3). Thus, FMR1 is an ideal candidate for chimeric construction in determining incompatibility domain in MAT A - l . The two chimeric genes, FMR1/A1 (with aa 1-92 of FMR1 and aa 93-end of MAT A-l) and A1/FMR1 (with aa 1-92 of MAT A - l and aa 93-end of FMR1), were used in the transformation reduction assay. Results (Figure 2-7) show that whereas FMR1/A1 (which contains the MAT A - l first LRR) confers incompatibility with a, A1/FMR1 (which contains the FMR1 first LRR) does not. Since both chimeric genes confer male mating activity in N. crassa, the lack of incompatibility in A1/FMR1 is unlikely to be due to global misfolding of the polypeptide. A reasonable explanation for the data is that the first LRR in MAT A - l is functionally important in mediating mating-type incompatibility. As shown in Figure 2-7, the incompatibility function of MAT A - l , as tested in various deletion, frameshift and chimeric constructs, is correlated with the presence of its first LRR.  Site-directed mutagenesis of the LRR and testing of another Neurospora mat A-l gene did not yield further information on which amino acid residue within the LRR is required for mediating incompatibility (see section 2.3.4 and 2.3.5). For example, the L109T (as shown in the site-directed mutant) and S95A (as shown in the N. pannonica mat A-l) change cannot be solely responsible for the lack of incompatibility function in the FMR1 protein. Since there are many differences between MAT A - l and FMR1 polypeptide within the first LRR region, further chimeric construction and site-directed mutagenesis are required to delineate the amino acids required for the incompatibility function. It is noteworthy to point out that the first LRR in MAT 82  A - l is predicted to form a (3-sheet (Chou & Fasman, 1978), whereas in FMR1, only the last three-quarters of the LRR is predicted to form the same secondary structure. The amino-acid difference in the first-quarter of the LRR (position 89-97; Figure 2-4A) may account for the difference in incompatibility activity of the two proteins.  N. pannonica is a homothallic species (Krug & Khan, 1991); all four mating type genes ace present and appear to be linked (L. Wheeler & N. L. Glass, personal communication). Since both mat A-l and mat a-l are present in the vegetative cells, the presence of mat A-l gene containing mating-type incompatibility suggested there may be atoZ-likesuppression in N. pannonica such that mating-type incompatibility is tolerated (see Chapter 4).  The MAT A - l protein has different functions in N. crassa and the results from this study suggest that they are mediated by different domains and probably by different biochemical mechanisms. MAT A - l is required for the mating event. The first 227 amino acids are required for male mating while the first 85 amino acids are required for female mating (see Figure 2-7). In comparison with the yeast system, the MAT A - l protein is probably a transcriptional regulator that controls mating-type specific expression of pheromone and receptors. MAT A - l (probably in cooperation with other transcriptional activator) may bind to the promoter of the A pheromone and the a pheromone-receptor genes and activate their expression. This activation activity is suggested to be mediated by the ct-domain (Hagen et al., 1993; Yuan et al., 1993) (see section 1.3).  From the result of this study, the incompatibility function of MAT A - l is likely to be mediated by its first LRR. LRR is a protein-protein interaction domain (Kobe & Deisenhofer, 1994). It is possible that this motif may mediate protein-protein interaction between MAT A - l and MAT a-l or between MAT A - l and TOL (or its downstream products). Recent yeast 2hybrid data showed MAT A - l and MAT a-l physically interact with each other (C. Staben, 83  unpublished results). Moreover, the mat a-1 A216-220 mutation (a 5-amino acid deletion within the first LRR of MAT a-1), which affects heterokaryon incompatibility without affecting mating activity and DNA binding in vitro (Philley & Staben, 1994), also destroys MAT a-1 ability to interact with MAT A - l (C. Staben, unpublished results). From the above data, the biochemical basis of mating-type incompatibility can be explained by physical interaction of MAT A - l and MAT a-1 and this interaction is mediated by LRRs. In P. anserina, a pseudohomothallic species, opposite mating-type nuclei exist within the same cytoplasm upon germination and mating-type incompatibility does not exist. Interestingly, an interaction between FMR1 (MAT A - l homolog) and FPR1 (MAT a-1 homolog) has not been detected via yeast two-hybrid system (Coppin et al., 1997). The MAT A-l/MAT a-1 dimerization model, however, does not explain how incompatibility is triggered. It is possible that the heterodimer binds to the promoter region of tol and regulates its expression; expression of tol would then trigger the incompatibility response, possibly through a signal transduction pathway. Alternatively, the two mating-type proteins may form multimers with TOL and that complex may in turn trigger the incompatibility reaction. These models cannot be fully tested until the components mediating incompatibility such as TOL have been characterized.  The MAT A - l and MAT a-1 proteins are multi-functional. Other than conferring mating and heterokaryon incompatibility function, MAT A - l and MAT a-1 are required for postfertilization events. The a  ml  mutant (with frameshift mutation in mat a-1) does not enter the  ascogonium or participate in meiosis (Perkins 1984; Raju, 1992b). Ectopic matA-1 copies can restore mating activity but not ascospore formation (Glass et al., 1988). In heterothallic and even some homothallic species, alternate mating-type nuclei are differentially recognized and paired during migration of nuclei into ascogenous dikaryotic hyphae (Shiu & Glass, 2000). Nuclear recognition is thought to be mediated by mating-type proteins (see section 1.3.4). The Cterminal part of both MAT A - l and FMR-1 is relatively acidic. In FMR-1, the C-terminal portion, which interacts with SMR2, is not required for mating but is essential for post84  fertilization function (R. Debuchy, personal communication). By analogy, MAT A - l may interact with MAT A-3 through its C-terminal tail, possibly to mediate its proposed postfertilization function (although the yeast two-hybrid system has failed to detect interaction between the two proteins; C. Staben, personal communication). Future experiments (e.g. through chimeric construction and mutagenesis) will give a finer delineation of the molecular basis of how the matA-1 gene mediates mating, incompatibility and post-fertilization functions.  85  3. Molecular Cloning and Sequencing of tol  (tolerant)  Some results from this chapter are published in Shiu & Glass (1999).  3.1 INTRODUCTION In addition to its role during the sexual cycle, the mating-type locus in N. crassa has a function during vegetative growth. Although fusion of opposite mating-type reproductive structures is required for entry into the sexual cycle, fusion of opposite mating-type hyphae during vegetative growth results in growth inhibition and cell death (Beadle and Coonradt, 1944; Garnjobst, 1953). A t least 10 additional genetic determinants called het (for Zieterokaryon incompatibility) loci also restrict formation of vegetative heterokaryons; genetic differences at these loci are not required for fertilization nor do they interfere with sexual reproduction (For review see Glass & Kuldau, 1992; Leslie, 1993).  The mat A and mat a sequences have been characterized and are composed of dissimilar sequences (Glass et al, 1988). The mat A-l and mat a-l genes are responsible for mating and heterokaryon incompatibility while the mat A-2 and mat A-3 genes are required for postfertilization functions (Glass et al, 1990; Staben & Yanofsky, 1990; Ferreira et al, 1996, 1998). A recessive mutation unlinked to the mating-type locus, tol (for to/erant), suppresses mating-type associated heterokaryon incompatibility such that tol A and tol a strains form a vigorous heterokaryon (Newmeyer, 1970). Mating activity of these strains is unaffected and tol strains o  show normal fertility in crosses with an opposite mating-type strain. Attempts to identify additional suppressors of mating-type associated incompatibility resulted only in the isolation of additional tol alleles (Vellani et al, 1994). The tol mutation apparently does not suppress incompatibility due to differences at other het loci (Newmeyer, 1970; Leslie and Yamashiro, 1997).  86  This chapter describes the molecular cloning and sequencing of the tol gene of N. crassa. The cloning of tol was made possible by the use of genetic mapping, chromosome walking and a functional assay.  3.2 MATERIALS AND  METHODS  3.2.1 Strains, media and culturing methods The N. crassa strains used in the study are listed in Table 3-1. Culturing and crossing, using V o g e l ' s (Vogel, 1964) and Westergaard's (Westergaard & Mitchell, 1947) media respectively, were performed as previously described, with modification (Davis and Deserres, 1970; Perkins, 1986). For heterokaryon tests, 2 p i conidial suspensions (10 /ml) of two strains 7  containing different auxotrophic markers were co-inoculated onto vegetative growth media (Vogel, 1964). A compatible heterokaryon forms a vigorously conidiating culture after 3 days of incubation with a mean growth rate of 7 cm/day. Mating-type incompatible heterokaryons are usually aconidial and have a growth rate of approximately 0.7 cm/day (Vellani et al., 1994).  3.2.2 Strain construction In order to introgress part of Mauriceville linkage group ( L G ) I V into T(IL—>IIR) 39311 ser-3A (Perkins & Barry, 1977; R L M 04-08, Table 3-1), R L M 04-08 was crossed with F G S C (Fungal Genetics Stock Center, Kansas City, K S ) strain 2226. (Mauriceville w i l d type a). Numerous restriction fragment length polymorphisms ( R F L P ) have been observed between Mauriceville strains ( F G S C 2225 and 2226; Table 3-1) and the Oak Ridge background of standard laboratory strains. Genomic D N A from ser-3 A progeny were screened for Mauriceville L G I V by hybridization to cosmid G 4 : A 9 (which contains trp-4, a L G I V locus closely linked to tol). A progeny, R5-28 (Table 3-1) had R F L P patterns identical to the F G S C 2226 parent when probed with G 4 : A 9 .  87  Genotype  Origin  his-5 A  F G S C 456  un-3 a  R . L . Metzenberg  tol trp-4 a  F G S C 2337  tol trp-4 pan-1 A  F G S C 4158  NE-1  his-5 tol trp-4 A  This study  M V lc-a  Mauriceville W T a  F G S C 2226  M V 1 c-A  Mauriceville W T A  F G S C 2225  RLM04-08  T(IL->IIR)39311 ser-3A  R . L . Metzenberg  R5-28  T(IL->IIR)39311 ser-3A  This study  Strain Rl-09  (Mauriceville background on L G I V ) R5-27  his-5 tol trp-4 un-3; a  This study  12-21-388  ad-3B al-2; cot-1; pan-2 A  A . J . F . Griffiths  R4-71  his-5 tol trp-4; pan-2 a  This study  R4-72  ad-3B; trp-4 cot-1; pan-2 A  This study  74-OR23-1VA  Oak Ridge W T A  F G S C 2489  1-20-41  ade-3B arg-1; tol A  A . J . F . Griffiths  1-20-26  ade-3B arg-1 A  A . J . F . Griffiths  I-10-1  ade-3A nic-2; tol a  A . J . F . Griffiths  1-1-51  ade-3A nic-2 a  A . J . F . Griffiths  OR8-la  Oak Ridge W T a  F G S C 988  Table 3-1. List of Neurospora crassa strains used i n this study. A l l strains are N. crassa Oak Ridge compatible (unless otherwise stated) and all tol alleles are N83. ( F G S C = Fungal Genetic Stock Center. W T = W i l d type.)  88  The strain N E - 1 (his-5 tol trp-4 A) was constructed by crossing his-5 A ( F G S C 456) with tol trp-4 a ( F G S C 2337); His" and Tip" progeny were selected. Crossing of N E - 1 to R l - 0 9 (un-3 a) gave R5-27 (un-3; his-5 tol trp-4 a). The presence of the tol' allele (N83) in R5-27 was confirmed by crossing with translocation strain R L M 04-08 (Table 3-1); approximately half of the A/a partial diploid progeny displayed normal growth rates (see Figure 3-2). The strain R5-27 was subsequently crossed with a pan-2 containing strain (12-21-388) to give pan-2 strains R4-71 and R4-72 (Table 3-1), which were used for transformation assays.  3.2.3 Chromosome walk and physical mapping A rA7?-4-containing cosmid was identified from the Orbach/Sachs p M O c o s X genomic library (Orbach, 1994; available from the F G S C ) using a trp-4 clone (Vollmer & Yanofsky, 1986) as a probe. A chromosome walk was performed in both directions from trp-4 using end fragments as probes for each step. Selected cosmids were physically mapped by restriction fragment length polymorphism ( R F L P ) analysis (Metzenberg et al, 1985).  3.2.4 Transformation assay and subcloning of tol Spheroplast preparation and transformation were performed as previously described (Schweizer et al., 1981; Vollmer & Yanofsky, 1986) with the exception of selecting for resistance to hygromycin B (250 units/ml; Calbiochem). Cosmids in the walk were assayed for T o f activity by co-transforming the cosmids (conferring hygromycin resistance) with a mat A-lcontaining p O K E 1 0 3 construct (pOKE103 has a pan-2 selectable marker; J. Grotelueschen and R . L . Metzenberg, unpublished results) into strain R4-71 (his-5 tol trp-4; pan-2 a) and strain R 4 72 (ade-3B; trp-4 cot-1; pan-2 A) spheroplasts. The tol gene was further subcloned into the hygromycin-resistant vector (pCB1004; Carroll et al, 1994). Homokaryotic transformants were isolated according to Ebbole & Sachs (1990).  89  3.2.5 Nucleic acid isolation and hybridization Standard molecular biology procedures were used throughout (Sambrook et al., 1989). Genomic DNA isolation from Neurospora was adapted from Oakley et al., (1987). Gel electrophoresis and nucleic acids transfer to Nylon filters (Schleicher and Schuell, Keene, NH) was performed according to manufacturer's specifications. [a- P] dCTP (Amersham, Oakville, 32  ON) labeled probes were generated from digested DNA using the T7 QuickPrime Kit (Pharmacia, Baie d'Urfe, PQ). The tol' mutant allele (N83) was amplified from strain R5-27 DNA using primers tol ATG1 (5'CCTGGGCTCACCTATGC 3'; base -50 to -34) and tol 3'-end (5' CGGCGGGATCTCTTTCTG 3'; base 2894 to 2877). The tol cDNA and tol (N83) PCR products were cloned into the PCRII vector using the TA cloning kit (Invitrogen, San Diego, CA) and subjected to DNA sequencing. The DNA sequence of two different tol' (N83) clones was determined and the mutation point was confirmed from three additional subclones.  3.2.6 DNA sequence analyses A 6.9-kb EcoRI tot containing construct was subcloned into an overlapping fragment suitable for DNA sequencing. DNA sequences were determined for both strands using the ABI automated Taq DyeDeoxy Terminator cycle method (Mississauga, ON) at the NAPS unit, Biotechnology Laboratory, University of British Columbia. Computer sequence analyses for protein and DNA were done using the MacVector/AssemblyLIGN software (International Biotechnologies, New Haven, CT) and GCG package available from the Wisconsin Genetics Computer Group (Devereux et al., 1984).  90  3.3 RESULTS 3.3.1 Isolation of a cosmid spanning the tol locus The tol locus is on the right arm o f linkage group ( L G ) I V and is flanked by trp-4 (~ 1 map unit) and his-5 (2-6 map units) (Figure 3-1). A cosmid containing trp-4 (G4:A9), was identified from a genomic library using the trp-4 gene (Vollmer & Yanofsky, 1986) as a probe. A chromosome walk was initiated from G 4 : A 9 in both directions. Twelve overlapping cosmids spanning a region of 350 kb around the trp-4 locus were isolated (Figure 3-1). The physical location of selected cosmids was determined using R F L P mapping (Metzenberg et al., 1985) and all mapped to trp-4 (Table 3-2).  To determine the orientation of the walk from trp-4, progeny that contained crossovers between his-5, tol and trp-4 from a cross between R5-27 (un-3; his-5 tol trp-4 a; Oak Ridge background) and M V l c - A ( F G S C 2225; Mauriceville background) were analyzed. Genomic D N A was isolated from H i s Trp" progeny and probed with cosmids shown in Figure 3-1. +  Recombination points in two his-5 tot trp-4 progeny (43-9 and 43-31) were identified when +  cosmid X 1 4 : C 2 was used as a probe (Figure 3-2A). These results indicated that cosmids directed toward G13:C8 were oriented towards the tol locus, and that the tol locus was centromere proximal to X 1 4 : C 2 (Figure 3-1).  91  — Q —  L  trp-4  tol  his-5 LGIVR  y ^  L  G4:A9 X14:C2  10 Kb  X4:E10 G6:E12 G1:E5  X17:B5 X10:A4 X17:C11 G6:C9  X7:C2 X25:D7 G13:C8  Figure 3-1. Contiguous cosmids isolated from a chromosome walk around trp-4 on the right arm of linkage group I V . The his-5 locus is centromere proximal (2-6 map units) to both the tol and trp-4 loci. A chromosome walk was initiated in both direction using the G 4 : A 9 cosmid, which contains the trp-4 gene. Orientation of the walk was determined by detection of genetic crossovers using cosmids as hybridization probe (Figure 3-2).  92  E  E  F  G  H  I  J  K  L  M  N  O  P  Q  R  13  57  13  14  57  68  14  14  14  58  23  24  14  24  1 4  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  mo  om  mo  mo mm  mm  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  -o  om  mo  mo mm  mo  mm  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  mo  om  mo  mo mm  m0  mo  mm  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  mo  om  mo  mo mm  X4:E10  m0  mo  mm  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  mo  om  mo  mo mm  G6:E12  mo  mo  mm  oo  mm  oo  oo  mm  om  oo  mo  mm mm  mm  mo  om  mo  mo mm  X2S:D7  m0  mo  mm  oo  m-  oo  oo  mm  om  oo  mo  mm mm  mm  mo  oo  mo  mo mm  G13:C8  mo  mo  mm  oo  m-  oo  oo  mm  om  ho  mo  mm mm  mm  mo  oo  mo  mo mm  A  B  C  D  1 4  67  1 4  G6:C9  mo  mo  mm  X17:C11  mo  mo  X10:A4  mo  trp-4  57  Table 3-2. Physical ( R F L P ) mapping of the trp-4 gene (in plasmid pSV50-31:2G) and cosmids identified i n the chromosome walk. Segregation of R F L P for 38 ordered progeny is detected by each clone i n the cross multicent-2a (Oak Ridge background or o, F G S C 4488) X M V l c - A (Mauriceville background or m, F G S C 2225). Progeny can be of either parental R F L P pattern (m or o) or be a hybrid (h). A l l cosmids identified i n the chromosome walk hybridization experiment (for the tol locus) is mapped closely to trp-4 in L G I V . (- = not scored)  93  3.3.2 Bracketing of tol by genetic crossovers and RFLP analysis To bracket the tol locus within the cosmid walk, it was necessary to detect recombination points that were centromere proximal to tol, i.e., between tol and his-5. A cross was performed between a R5-28 (T(IL->IIR) 39311 ser-3 A; M V L G I V ) x R5-27 (un-3; his-5 tol trp-4 a) (Figure 3-2B). The R5-28 strain contains an insertional translocation in which the left arm of L G I (which includes ser-3, un-3 and mat) was inserted into the right arm of linkage group II (Perkins & Barry, 1977). Progeny were selected for their ability to grow at wild-type rate at 30°C in medium lacking serine (selection for A/a tol partial diploid progeny; Figure 3-2B) and histidine but containing tryptophan (selection for progeny with recombination between his-5 and tol). Genomic D N A from sixty-eight Ala tol trp-4 progeny were screened for R F L P differences from the R5-27 parent when probed with the cosmids centromere proximal to G 4 : A 9 . Fifty-nine progeny contained R F L P s that were identical to the R5-27 parent, indicating that in these progeny the recombination point between his-5 and tol lay centromere proximal to the cosmids identified in the walk. In genomic D N A from eight progeny, a R5-28 pattern was identified when G13:C8 was used as a probe, but a R5-27 pattern was observed when probed with X 2 5 : D 7 (Figure 3-2B; Appendix 8.1). These data indicated that the recombination point between his-5 and tol in these progeny occurred between G13:C8 and X 2 5 : D 7 and that tol must be centromere distal to the crossover point. When genomic D N A from progeny T l 1 was probed with G13:C8, a hybrid R F L P pattern was identified, consistent with the interpretation that the tol locus resided between G13:C8 and X 1 4 : C 2 .  94  A his-5  _L_  tol  trp-4 his-5 tol trp-4  trp-4 (43-9, 43-31)  ov  MV  + X14:C2  Figure 3-2. The tol locus was bracketed by genetic crossovers and restriction fragment length polymorphism ( R F L P ) analysis of progeny. (A) Crossovers between tol and trp-4 were detected in the progeny 43-9 and 43-31 (his-5* tot trp-4) which displayed hybrid R F L P patterns that differed from both parental strains when probed with X 1 4 : C 2 .  95  B  R5-27  R5-28  ser-3+ un-3-  <  LG I  )  a  -o  O  un-3+ A ser-3'  LG II  O  O  his-5+  LGIV  tor>  trp-4+  his-5-  Qm  trp-4-  tol-  O  Select A/a partial duplication progeny  at 30° (un-3 ) with wild-type growth in +  medium with tryptophan ser-3+  un-3- a  LG I  o  un-3+  LG II  to/-  his-5+  LG IV  O - L -  trp-4-  1  mmJ G13:C8 X25:D7  T11  his-5+  Or  Qil  tor  1  trp-4-  1  G13:C8 X25:D7  T4,T5,T10,T15,T18, T21, T35 and T37  Figure 3-2. The tol locus was bracketed by genetic crossovers and restriction fragment  his-5 and tol was selected for by isolating A/a partial duplication progeny that were tol, trp-4 and his-5 . R F L P analysis of genomic D N A from A/a tol trp-4 progeny identified crossover length polymorphism ( R F L P ) analysis of progeny. (B) Recombination between +  points between cosmids G13:C8 and X 2 5 : D 7 . When probed with G 1 3 : C 8 , many progeny displayed the 39311 ( M V L G I V ) (T4, T 5 , T10, T15, T18, T 2 1 , T35 and T37) or hybrid pattern ( T i l ) (see Figure 8-1), indicating that the tol locus resided between G13:C8 and X 1 4 : C 2 i n the cosmid walk.  96  3.3.3 Transformation reduction assay for Tol activity +  The T o l activity of contiguous cosmids between G13:C8 and G 4 : A 9 was assayed by co+  transforming each cosmid with pOKEmat A-l into R4-71 (his-5 tol trp-4; pan-2 a) and R4-72 (ad-3B; trp-4 cot-1; pan-2 A) spheroplasts. Transformants were selected for both hygromycin resistance (cosmid marker) and for growth in the absence of panthothenic acid (pOKEmat A-l marker). Transformants fail to regenerate following the induction of mating-type associated incompatibility (Glass et al. 1990) and therefore a cosmid with T o l activity should exhibit +  significantly lower transformation frequencies when introduced with mat A-l into tol a spheroplasts as compared to A spheroplasts. Only cosmid X 2 5 : D 7 (plus mat A-l) exhibited a significant reduction in transformation frequency i n a tol a background. Further subcloning identified a 6.9 kb E c o R I fragment from X 2 5 : D 7 that caused a 20 - 30 fold reduction in transformation frequencies when introduced into tol a spheroplasts as compared to A spheroplasts (See Appendix 8.2).  3.3.4 Molecular characterization of tol sequence D N A sequence determination of a 4.2 kb Sall-Nsil sub-fragment from the 6.9 kb EcoRI fragment revealed an open reading frame (ORF) of 3127 bp interrupted by a putative intron of 94 bp. The 5' and 3' and internal sites of the tol intron fit intron splicing consensus sequences (Bruchez et al., 1993; Figure 3-3). The sequence of a tol c D N A spanning the intron site was characterized and confirmed the presence of the intron in the genomic tol sequence. D N A sequences surrounding the tol A T G start codon were i n good agreement with the consensus for N. crassa (Edelman & Staben, 1994). A sequence matching the transcription initiation consensus for N. crassa was found at position -324 ( T C A T C A N C ; Bruchez et al., 1993) and a C A A T motif (Bucher, 1990; Chen & Kinsey 1995) was identified at position -413, 85 bp away from the proposed transcription start site (Figure 3-3). Three pairs of short perfect repeats ( C G C C G C C C A , T T T G T T G , and G A G A A G T T C A ) were found 5' to the proposed C A A T box.  97  Translation of the tol O R F identified a 1011 amino acid (aa) polypeptide (Figure 3-3) with a calculated M of 113712. T O L has a predicted isoelectric point of 4.67 and is made up of r  40% non-polar and 26% charged residues. The carboxyl-terminal portion of T O L (amino-acids position 837 to 1011) is rather hydrophilic, composed of 30% non-polar and 30% charged residues (see Hydrophobicity Plot in Appendix 8.3). B L A S T (Altschul et al., 1990) and F A S T A algorithm (Pearson & Lipman, 1988) searches did not reveal significant similarity between T O L and any other protein sequence present in protein databases. However, a heptad repeat structure was identified from amino acid position 177-211 (Figure 3-4A and B ) , as predicted by the C O I L S program (Lupas et al., 1991). A coiled-coil is an a-helical bundle that is thought to wind into a superhelix, with the hydrophobic residues (a and d) forming the hydrophobic packing interface (for review see Lupas, 1996). Coiled-coil domains play structural roles in numerous fibrous proteins (Pauling & Corey, 1951; Crick, 1953) and are also a dimerization motif in the leucine-zipper class of transcription factors (Landschulz et al., 1988). The putative coiled-coil domain in T O L had five heptad repeats and contained mostly hydrophobic residues in position a and d with some charged residues in position b, c, e, f, and g. Occasional polar residues in the core (a and d) (such as the case in T O L ) favors dimerization over trimer- or tetramerization, since they can still be partly solvated (Harbury et ai, 1993; L u m b & K i m , 1995).  A putative leucine-rich repeat ( L R R ) was found in the carboxyl-terminal portion of T O L (position 804-823; Figure 3-4A). L R R s , which have been found in numerous proteins, are thought to mediate protein-protein interactions (For reviews, see Buchanan & Gay, 1996; Kobe & Deisenhofer, 1994). Each L R R contributes an exposed P-sheet that could participate in strong interactions with other proteins. The L R R in T O L is predicted to form a P-sheet by both the Chou-Fasman (Chou and Fasman, 1978) and the Robson-Garnier algorithms (Gamier et al., 1978). Although L R R s are usually present in tandem arrays, at least three proteins with a single L R R have been reported; Rev (HIV-1 nuclear regulatory protein; M a l i m et al., 1989), GPIbp (Human platelet glycoprotein lb p subunit; Lopez et al., 1988) and G P I X (Human platelet  98  -818 -800  GAGCAGCAGAAGGTCTGGACTGClAAGGCGCCnXCAACGAA^  -700  GTCGACACGCAGTTCCCT  TTTgrKXX3GACACGGAflCACTCGGAAATCG^  TCCGGCGAGCACCAGCTITCTGGCGIAGGGCTITGTGG^  -600 -500 -400  AAGCGGAAGCAGTGAGTAGTCTGGGGCCCTTAGATAACAGTAAA^ CreOYrACATGAAGAAGTGTGTACCGAAATCA^ CAGTATGTCAATCCTACAGAAAGTAGGAGGATAGATGTTAGT^  -300  CCCCAAGGGAAAACGAGCACTTACCTAOSAACCCACAGT^^  -200 -100  TACTACGCTATGCTACACTACAGCTGACGACAACGTCG^ (XTICCSGAAAGTCTITCGACTITAC^^ 1  101 201 301  401 501  601 701  ATGATATCCCCATCCTCCAGCTGCCCAACATCTICT M I S P S S S C P T S S I N S N T Y P V S S S S A S S P I P S S S ATCTAGATCCCGAATCAAGATCCCACAAAGGATCC^^ H L D P E S R S H K G S Y E A V L E Y F T S N S L P L P S Y L Q T TGACTCTITCGTCTCTCACT^ D S F V S H F F T S T D N P S L L S P I N I P P H L T S Y E A F I CXJGTATICCATCGCCAAAGCAGCCCCTGCAGCATCC?IX^CTO R Y S I A K A A P A A S S L P S T V P L P L N Q F C P R C T K A L  TGGAGGGATGTCAAGTATCTCCTGGTK^CT^^  R  R  G  G  E  R  R  L  V  V  L G  W R D V K Y L L V G } L S F W A E GAGTGCCICTGGGAAGGGAGAAGGATGOGGAGGTGGATATCGAGG^  R  V  P  L  G  R  E  K  D  V  D  V  D  M  E  G  C  K  T  V  P  Q  D  R  R  V  V  V  E  I  E  E  E  D  D  S  L  F  E  V  R  E  W  S  D  R  A  L  G  S  K  E  E  G  Q  V  Q  K  S  V  V  E  F  V  R  L  R  R  G  Y  E  W  I  N  N  C  R  S  H  C E  C  I  P  P  S  F  L  A  S  F  H  T  A  S  GCTGCCGAGACCATGGACTGTCACGAACGGCGTCCT A  A  E  T  M  D  C  H  E  R  R  P  E  TGGCTGTITCCXSAGACACCCTTCATCTC^ L  A  V  E  G  S  I  E  R  T  P  L  F  T  M  E  S  L  P  Q  <A  GGAAGGTATCAGGCTTACCGAGCTGCCCAAGAC^^  S  R  W L  L  S  H  C  W  K  T  F  R  D  K  1401  C I I Q D S K E D W D D E S V CCAACTCTCACGGCGGGCTX^TICAACCCGCGCCATCCGCT^  P  T  1801  1901 2001  2101 2201  2301 2401 2501  2601 2701 2801 2901  3001 3101  S  H  G  G  L  F  N  P  R  H  P  L  S  Q  A  G A  TGCATTATCCAAGACTCTAAAGAAGACTGGGACGACGAGTCGGT^ N  S  I  1301  1701  7  V  V  200  P  233  P  265 S  P  269  Y  TCCCCCCICCITCCTCOXTCCI^^  1201  1601  6  QZGCCCAGAAACIUIKTTAAAAGATttxACTIGAACAGTC^  1101  1501  133  1  E  R  G 1001  67 100  CATCATCCCCACCACCCCOTraaOX^ A S S P P P P S H A A W W TCAATICIXXTGGTTTGTGATGGTGAAGGTG^ Q F C W F V M V { K V L  801  901  33 Y  V  K  D  I  V  GAAGCTGGGGTITCCAGAGGCCGAGriGTGTGGATGGC^^  T P  P P  I  E  V  P  L  S  D  D  N  D  L  F  T  R  G  W  V  S  F  P  S  A G  T  D  T  G  N  K  S  R  S  L  T  K  A  S  L  A  G  L  W  S  W  A  S  GAGCGAACGAGATCAGTITCGGATITrAGGA^  Q  R  E  T  W  A  S  V  N  R  S  Y  L T  R  V P  D  L  P V  G  V  E R  N  C  G  V T  E  N V  P Y  535 569  I  W  602  Y  635  D  L  P  L  502  S  A  G  D  L  D  T  F  469  E  I  V  402  Y  Q  L  G  369  435  D  A  L  K  S  R  P  335  L  A  E  L  P  T S  D  R  G  K  Q  G  D Q  H S  L  F  G  I  302  A  D  D  L S  S  T  I A  E  C  R  W A  I  K L A F A E A E C V D G L A E V L E GTICCTGGTTCTGGGAGTATCAGGCTTAQGGGGCOGGTGCTGC^ V P G S G S I R L R G P V L P R P R I  D  I L  C  F  Y  Y  L E  N  L  Y  N  P  I  L  T  S  G  V  V  V  D  Y  K  I  R  S  S  T  L  N  V  N  V  L  L  L  H  R  S  N  T  A  Y  E  E  E Q  R  V  W  G T  Q  C  TCOOTATAGCAGAGCAACTCAGCGGTCGATGGGGCCCG^  V  V  Y  P  GCTGGGACGGCGATATGiAATGACAQGCGAACCATCTITCAGCATCAGTC  A  R  T  Q  R W D G D M N D R R T I F Q H Q W TAGTGACATGAACIXXAAGOGGCGCAAAGCCTGGGAACTCCT S D M N S K R R K A W E L L > V Q S G I A E Q L S G R W G P G TGGGCGGCGAAGATCGGTCTIGAGGAGCGGG!AT03XAQGC M G G E D R C E E R D A Q A  G  V  M  L  G  E  CAACATCAAGAATAAGACGTICCTAACAAGCGAGTACOGCTCCGAA N I K N K T F L T S E Y R S E K CTCGCCCGGOGCACGCTAATCTIOSGCAAGGAAGAACTGC^ L A R R T L I F G K E E L H W E  A  669  V  V  702  F  735  G A N E M S S D F R M S E S G V Y V N M A T G M P A Y L Q S Y Q S I CGGGGTGCX7ITCGGACAATATCAATCATATGAAGGAAGATG A V R W D N I N D M K E D E E E A Y L A P L Q V Q L I E D G P A G AACCCGAGCAAACTTCAACTTCTCGGATIGGT^ N [ P S K L Q L V G L V L L K A S S W V A V ] P Q F R R V G L F F I T  AOGAGGCACCTCAGTGGAACTCTGCCCCTGACAA^ D E A P Q W N S A P D N E L CTCTraSGAOXGGAATCTIGaXra^ S  S  D  P  E  S  C  A  V  R  Y  P  GACATCTGTICCGAAGATIOTACrc&T^  Q  D  D  P  Q  R  D  N  .  E  Y  G  H  H  H  P  P  D  T  A  T  W  E  S  E  S  N  D I C S E D S Y S S N P F A D AGGAATACTATTAOGAAGACTICTCCGTCTCGGCATC^ N E Y Y Y E D F S V W A S G I E  R  AATICAGCAAGGGCAACGATCGGATCGCTIGAAGGAA^ I  Q  Q  G  Q  R  S  D  R  S  N  *  L  TCTIXAACGGAOJITFCrACCATCGAAT^^ S  P  T  D  V  L  P  K  E  D  P  E  Y  3201 3301  AATGGTTATTATGTITATGAGATTGrrCTAAAGGra CXIATACAGCAGTAAACAGCCTAACGTCCAACACTGGTATGAGC^  3401  TGCAT  L A  K N  N G  Q P  G R  V P  G F L  T  A  D E R N  T  D E N T  H  D T  S  M  N  V  D  F  E  S  N  S  Q  P  D  E  R  D  P  I  D  T  W  F  N  L  A  T  R  A A Y  S  S  802 835  8  6  9 902  E Q  769  F E  935 9  T  6  9  V  1002 1011  Figure 3-3. Nucleotide and amino acid sequence of the tot gene of N. crassa. The nucleotide sequence (4.2-kb Sall-Nsil fragment) and the predicted T O L protein sequence are shown. The intron sequence is italicized and the 5', 3' and internal splicing consensus sequences are underlined. Sequences matching the consensus for a transcriptional start site and C A A T box are underlined in bold. Short repetitive sequences (3 pairs) in the promoter region are underlined. The putative coiled-coil domain, the leucine-rich repeat ( L R R ) and the S E T domain (a region of homology between H E T - S I X , H E T - E and T O L ) are bracketed with {}, [] and <>, respectively. The original tol mutation, E858 ( G A A ) —> Stop ( T A A ) is underlined in bold and italicized. The GenBank accession number for this sequence is AF085183.  99  TOL  N  177-211  315-583  HZ  804 - 823  1011 aa  SET PSKLQ LVGLVLliKASSWVA V  todefgatedefgatedefgaJDaaefgabc^fga Coiled-coil  PXXaXXLXiaxxijXaXXXaXXa LRR  B RIKAL VGEEEK  V E W L  PQLVL  VDKDG  LVDEY  Figure 3-4. Structural features identified in T O L . (A) The amino acid sequence of the coiled-coil domain (position 177-211) in T O L is shown with a schematic heptad position a-g underneath. The leucine-rich repeat ( L R R ; amino-acid position 804-823) is shown and the consensus for Arabidopsis thaliana RPS2 L R R (Mindrinos et al, 1994) is shown underneath. For the L R R , an " X " stands for an arbitrary amino acid while an "a" stands for an aliphatic amino acid. The S E T domain (position 315-583) represents a region of homology between H E T - S I X , H E T - E and T O L (see figure 3-5). (B) A helical wheel representation of the coiled-coil domain in T O L . The hydrophobic residues of the heptad repeats are in bold while charged residues are underlined.  100  glycoprotein I X ; Hickey et al., 1989). These single L R R s are thought to be functionally important ( M a l i m et al., 1989; Noris et al., 1997).  A region of similarity has been detected between T O L and the predicted products of the het-6 locus of N. crassa and het-e locus in P. anserina (Smith et al., 2000b) (Figure 3-5). This region, hereafter referred to as the S E T domain ( H E T - S I X , H E T - E and T O L ) , corresponds to several conserved stretches of amino acids that span 269-aa in T O L (from position 315 to 583). This region is distinct from the GTP-binding domain and the (3-transducin-like W D 4 0 repeats in H E T - E and the L R R and coiled-coil domains in T O L (see section 1.4), and thus may represent a domain necessary for vegetative incompatibility that is in common between all three het interactions.  A search on the database in the Neurospora Genome Project (Munich Information Center for Protein Sequences; http://www.mips.biochem.mpg.de/) revealed two O R F s located on L G II that encode predicted polypeptides with similarity to T O L . Predicted polypeptide from clone B 3 E 4 contains sequence simlarity with T O L from amino-acid position 310 to 658 (p-value = 3 x 10" ), which spans the S E T domain. Predicted polypeptide from clone B 1 4 D 6 has similarity 25  with T O L from position 119 to 635 (p-value = 1.4 x 10" ), which spans the coiled-coil domain 22  and the S E T domain; the coiled-coil domain is not conserved in clone B 1 4 D 6 . The two clones do not have significant D N A identity with tol.  3.3.5 Complementation of the tol mutant and DNA sequence analysis of the tol" allele The original tol mutation was identified in an A/a partial diploid strain that had escaped from inhibited to wild-type growth rates (Newmeyer, 1970). The mutation was subsequently mapped to L G I V , near trp-4. T o confirm that the transformation reduction result is not an artifact and that the tol gene was cloned, complementation experiments with the tol' mutant was performed and the D N A sequence of the tol ~ allele was determined (N83).  101  6 6 H T  PA . * O R . . TB . . O L . 3  fi  PA 6 OR HTB TOL  6 PA OR HTB TOL  fi  HPffiS S Y T D D L S G Y I HPBSCYTDDLYCCI jRDD|aEIRP T AfflGR P G K T H V R - V I B T  3 0 3 0 .1 15  A  v  LKiSvQ-  p  I  PA OR HTE TOL  S  G Y F ABBJE K R P KRJDjG N R I BH S F T g g H V E R T H E WKWABQKCK^ KTF0TS BYR S B | E S D L I L  Q S Y K V V Y A • AY KV I Y M  IN--  P  YH  DD AD K S S Q M L L 3 K M i 3s s DNBD KS F Q K R L 9 R D | flss DKSNSTp LQBAflNS MPR W 3RD I p S K E K W D D E SVK  2  H  SJRN  I  E Y L L S V R R L I B K N I B Q P O V V A E H W L S V D R L I K E K I B Q P D A V T  p._a  K D Y N  F cfflCS DEjyFjAC G Y K V V P E H F V S | V T  FCQCADHJHACGYKVVSQ0MVS|LT  L I A P T S VEHJFS QLHARRHII FO  R E Q A R IffiDR KEELHW  M Q K C L R E R L B T P E B Q T Y C I L V g O Y L SHHLSFraa'RVHC Q Y P N A K M T S E H L I M H K C L R S l L I I P D B Q T Y S T LFESO L M S B F P I F H H J R G Y C O Y P Y R K B T I E H Lj IQ v E B N T T R E @ D H A Y SSQFOB [ R H H S v T G I P M E A L R G l B - - L S D F @ V H D J I l A w f K0|f W B K L T Q T D T O N K SAS ISFPSSBDRERWDQ DMND J R T I F QHQ  6 PA OR HTB TOL  fi  60R HTE TOL  E @ P KTFfJ[DA lEfflT Q R  L  QVSWSHtjP A A D G S D K L - - M D A F V E | O Q Q P L D K | a j K T T Q E0IfllI A V D D S N R V - - M D A L A E S G Q E F L D K | | G jHTE K C YH]|QT D V S T D K R jADG L N B A A G | S P N S H G G L F N P R H P L S T ^ P W S I E V P U S H D N D  fi fi  6 PA  B*|B*  V T F J J J Q T S Q D T M L Q H L R T I i Q H Q wfiSs Q T pfyWIs sfflp T M L M H L R B M H Y R R M I E B L P! S K H Q Y H - R BR F Q A DQAWRDORKFF  RgSllWl-  STRTHraiHVVNlWjjjj • jGgF D S T R T B | I B V A N H Q j -QP PDE H E V S Y K r a K l a S i - 0V K DO V PJJJQL L K G E J Y D L J F I X  YT$|gggO.A j SQ|!A Y T X p Q P I j SgJQA KDpJsGKI j P | l I AO *H-BH*  H  A G S T R F Y A T N Q  02H S°IE V  HvGVTPPCVTNKnraJvBoi!' 0D V H L P L I Y G E G K K C A L E R j L O P A D S SDMN SKHRQABEBE  Figure 3-5. Sequence similarity among T O L and the predicted products of two het genes, het-6 of N. crassa (denoted by 6 0 R and 6 P A ) and het-e of P. anserina (denoted by H T E ) .  Black  boxed illustrate amino-acids identity while grey boxes represent amino-acids similarity. This region of homology w i l l be referred to as the S E T domain ( H E T - S I X , H E T - E , and T O L ) . Reprinted from Saupe (2000).  102  A p O K E 1 0 3 - t o / construct containing the 6.9 K b EcoKL-EcoRl fragment (pOKE-EE6.9) +  (Figure 3-5) and a vector control (pOKE103) were transformed into tol a spheroplasts (strain R 4 71). Homokaryotic pan-2 tol a (pOKE103 and p O K E - E E 6 . 9 ) transformants were isolated and +  subjected to heterokaryon tests with toT A (1-20-26) and tol A (1-20-41) testers. A s expected, homokaryotic pan-2 tol a transformants containing only the vector (pOKE103) formed a +  vigorous heterokaryon with the tol A (1-20-41) strain, but formed an incompatible heterokaryon with the tol A tester (1-20-26). In contrast, the pan-2 tol a ( p O K E - E E 6 . 9 ) transformants +  +  exhibited typical mating type associated incompatibility with both tol A and tol A testers. These +  data indicated the original tol mutant phenotype was reverted to wild-type (Tol ) by the +  introduction of the p O K E - E E 6 . 9 clone. T o confirm further that tol was cloned, the D N A sequence of the tol' mutant allele was determined. The tol" allele (N83) contained a transversion mutation at position 2666 (G—>T) that resulted in the change of a glutamic-acid codon ( G A A ) into a stop codon ( T A A ) at amino acid 858 (Figure 3-3).  3.4  DISCUSSION This chapter describes the molecular cloning and D N A sequence analysis of tol, a  mediator of mating-type associated incompatibility in N. crassa. The tol locus is the only molecularly characterized mediator of allelic incompatibility from fungi. Extragenic suppressors for allelic incompatibility loci such as N. crassa het-c (N. L . Glass, unpublished data) and P. anserina het-s (cited in Coustou et ai, 1999) have been isolated but they have not been molecularly characterized (see section 1.4). Non-allelic suppressors of incompatibility have been characterized in P. anserina and encode proteins thought to be involved in developmental processes and signal transduction (Loubradou etal., 1997, 1999; Barreau etal, 1998) (see section 1.4).  103  Since heterokaryon incompatibility is a phenomenon leading to hyphal compartmentation and death, it is impossible to isolate components of the het system by virtue of positive screening using functional complementation. A combination of genetic mapping, chromosome walking and functional assays was utilized to clone the tol locus. The het-c and het-6 loci of N. crassa were cloned using similar methods (Saupe et al., 1996a; Smith et al., 2000b), whereas het-c, hete and het-s of P. anserina were isolated using a combination of SIB selection and phenotypic expression (Saupe et al., 1994; Saupe et al., 1995a; Turcq et al., 1990) (see section 1.4). Mutants in modifier (mod) genes of P. anserina have phenotypes (sexual and/or vegetative) other than compatibility which allows their cloning by complementation (Loubradou et ai, 1997, 1999; Barreau et al, 1998).  The Tol activity (and hence the tol ORF) was identified by a transformation reduction +  assay utilizing the fact that co-transforming the mat A-l and tot genes into a strain with tol a background induces cell death and hence reduces transformation frequencies. The successful cloning of the tol gene was confirmed by complementation of the tot mutant and the sequencing of the tot alleles. The predicted TOL polypeptide was not identified as a functional homolog of any other proteins found in the genome database. However, it contains two protein-protein interaction domains, the leucine-rich repeat (LRR) and coiled-coil domain. A LRR is implicated in the mediation of incompatibility in MAT A - l (see Chapter 2). Therefore, protein-protein interaction between TOL and other protein(s), such as MAT A - l , could be an important factor in mating-type incompatibility. A region of homology is found among TOL, HET-6 of N. crassa and HET-E of P. anserina (the SET domain; Figure 3-5). HET-6 and HET-E are both het genes involved in mediating non-allelic incompatibility (Smith et al, 2000b; Saupe et al, 1995a). The HET-E polypeptide contain a GTP-binding domain and a WD40-repeats (as found in all GP proteins) whereas HET-6 does not contain any identifiable functional motif. The GTP-binding activity and the number of WD40 repeats are crucial for HET-E function (Saupe et al, 1995a; Espagne et al, 1997). The region of similarity (a span of 269-aa in the case for TOL) is distinct 104  from the LRR, WD40, coiled-coil and GTP-binding domain. This is the first time that sequence similarity between gene products of different het systems has been reported. Saupe (2000) has raised the possibility that this region of homology may represent some sort of "death domain controlling common downstream targets". Although it remains to be determined whether this region may represent a functional domain of any importance, the similarity provides encouraging evidence that different het systems may share a common biochemical pathway.  Two ORFs that encode a predicted polypeptides similar to TOL have been discovered on LG II. It is not clear if the two ORFs are expressed and what their function might be. It is possible that they are related to tol and that they may be of the same gene family. Future study of the twotoMikeORFs may give us hints on the evolution of tol and how tol mediates matingtype-associated incompatibility. Since the region of similarity between TOL and the two ORFs spans the SET domain, they may confer function for heterokaryon incompatibility, or even represent unidentified het loci in N. crassa. Further investigation is required to determine if thesefo/-likesequences have any role in heterokaryon incompatibility.  Some het genes have cellular functions other than to limit heterokaryosis (For review, see section 1.4). The original tol mutant shows normal vegetative growth and sexual reproduction. However, the tol mutation has been shown to suppress the phenotype of afinf-1mutant (Johnson, 1979), which is sterile as both a male and a female in crosses to a wild-type partner. Fertility is restored when a fmf-1 tol strain is crossed to a fmf-l tol strain. It is not clear how mutations in +  tol, which do not affect sexual development, can suppress the fertility defect of fmf-1.  Some species within the genus Neurospora do not exhibit mating-type-associated incompatibility. This fact is presumably due to the presence of tol mutations within these species that suppress mating-type-associated incompatibility. In the heterothallic species N. sitophila, isogenic strains that differ only in mating-type will form a vigorous heterokaryon. However, 105  when mat A and mat a from N. sitophila were introgressed into N. crassa, mating-type associated incompatibility was observed (Perkins, 1977). In the pseudohomothallic species, N. tetrasperma, opposite-mating type nuclei normally reside in a common cytoplasm. Similar to the results observed with TV. sitophila, mat A and mat a from N. tetrasperma displayed mating-type incompatibility when introgressed into N. crassa (Metzenberg and Ahlgren, 1973). The introgression of tot from /V. crassa into N. tetrasperma induced mating-type associated incompatibility and disrupted the pseudohomothallic nature of the species (Jacobson, 1992). A l l of these data, and the fact that all suppressors for mating-type incompatibility that have been isolated are allelic to tol (Vellani et al., 1994), argue that tol is the major mediator of mating-type associated incompatibility. The evolutionary history and selection for or against mating-type associated incompatibility in the different species within the genus Neurospora remains an enigma. The molecular characterization of tol from N. crassa and related species w i l l provide the necessary tools to address this question.  It is believed that heterokaryon incompatibility is a universal phenomenon among filamentous ascomycetes and basidiomycetes (Glass & Kuldau, 1992; see section 1.4). It has been proposed that vegetative incompatibility may protect an individual from the transfer of deleterious cytoplasmic factors (Caten, 1972). Cytoplasmic transfer of hypovirulence-associated d s R N A virus in Cryphonectria parasitica and detrimental K a l D N A plasmids in N. crassa are reduced by vegetative incompatibility (Anagnostakis, 1982a; Debets et al., 1994). In black Aspergilli, vegetative incompatibility completely blocks transfer of mycoviruses (Van Diepeningen et ah, 1997). Vegetative incompatibility mediated by mating-type could be selected for by two mechanisms. The first mechanism is that incompatibility mediated by mating type could promote outbreeding by eliminating the possibility of heterokaryon formation with opposite mating-type siblings. However, in a outbreeding population, the formation of vegetative heterokaryons is also excluded by differences at any of 10 additional het loci in N. crassa; N. crassa populations are highly polymorphic for het loci ( M y l y k , 1976a). A second  106  i  mechanism is that matA-1 and mat a-1 evolved solely for sexual reproduction, but that molecular divergence of tol resulted in mating-type associated incompatibility during vegetative growth. Since the mating-type locus is always polymorphic in populations in a heterothallic species like N. crassa, mating-type associated incompatibility would be an efficient way to restrict heterokaryon formation with 50% of the population, even in the absence of polymorphisms at other het loci.  Mating-type-associated incompatibility has been reported in other fungal species, such as in Ascobolus stercorarius (Bistis, 1994), Aspergillus heterothallicus ( K w o n & Raper, 1967) and Sordaria brevicollis (J. Bond, personal communication). It is possible that the phenomenon of mating-type associated incompatibility is even more widespread i n filamentous ascomycetes, but cannot be assessed because of the lack of isogenic strains that differ only at the mating-type locus. The characterization of the three major components of mating-type associated incompatibility, matA-1, mat a-1 and tol, w i l l provide tools to analyze the molecular mechanism of mating-type associated incompatibility in filamentous fungi and w i l l facilitate the challenge of delineating the molecular mechanism of growth inhibition and cell death that is a characteristic feature of this phenomenon.  107  4. Characterization of tol and Isolation of top (TOL-interacting Protein) Genes Some results from this chapter are published in Shiu & Glass (1999).  4.1  INTRODUCTION The cloning and sequencing o f tol is described in chapter 3 o f this thesis. The tol gene  encodes a putative 1011-amino acid polypeptide with a coiled-coil domain and a leucine-rich repeat. Characterization of mutant alleles in chapter 2 demonstrates that a leucine-rich repeat is required for incompatibility function in mat A-l.  Attempts were made in this study to identify  region(s) o f the T O L protein required for mediating mating-type-associated incompatibility.  Since the mutation in the original tol" strain (allele N83) occurred at the 3' end of tol (near the carboxyl-terminus of T O L ) , it is possible that the N83 mutant is not a to/-null mutant and may retain partial function. Attempts were made to obtain additional tol mutants by repeat induced point (RIP) mutation (Selker, 1990) and to analyze their phenotypes. R I P is a mechanism in Neurospora that causes duplicated sequences to undergo multiple G - C to A - T transition mutations during sexual reproduction. The R I P phenomenon is utilized as a means to introduce mutations i n a gene in vivo. A given gene can be mutagenized by transformation o f a copy of that gene into a recipient and taking the transformant through a cross. Before karyogamy, but during mitotic divisions within ascogenous hyphae, the resident as well as the introduced ectopic copy undergo GC—»AT transition mutations. The frequency of the mutations range from 10-70%, depending on the gene used, the insertion site, and the size o f the fragment introduced (Irelan et al., 1994).  108  One enigma of mating-type associated incompatibility is the nature of relationship between mating-type proteins and T O L . One possibility is that tol is a downstream effector of the incompatibility pathway whose expression is regulated by the mating-type proteins. Expression patterns of tol were analyzed in this study in order to shed light on plausible mechanism of interactions between mating-type proteins and tol.  The mating-type genes have seemingly opposite functions in different stages of development. Although the mating-type of two isolates must differ for sexual development to occur, the alternative alleles trigger incompatibility during vegetative growth. Since mating-type proteins are required after fertilization, presumably for nuclear recognition during proliferation and partitioning of opposite mating-type nuclei in the crozier (Shiu & Glass, 2000), the incompatibility reaction between antagonistic matA-1 and mat a-1 genes must be suppressed during sexual development. The possibility of differential regulation of tol during sexual development was also examined in this study.  Vegetative incompatibility between opposite mating-types is due to the molecular actions of M A T A - l , M A T a-1 and T O L . The M A T A - l and M A T a-1 gene products are transcriptional regulators while the molecular function of T O L is not apparent, although it contains putative protein-protein interaction domains. T o understand the role of T O L in mating-type heterokaryon incompatibility and to identify other proteins involved in this process, several putative T O L interacting proteins (top) were identified using the yeast 2-hybrid system. The yeast 2-hybrid system detects protein-protein interactions in vivo (Fields & Song, 1989). It utilizes the property of eukaryotic transcriptional activators, which consist of two separable domains - the D N A binding domain ( B D ) and the transcriptional activation domain ( A D ) . When a protein X , which is fused with B D , interacts with a protein Y , which is fused with A D , the transcriptional activator is reconstituted and transcription of reporter genes is therefore activated.  109  Many Neurospora species do not exhibit mating-type incompatibility. Examples are the heterothallic N. sitophila and pseudohomothallic N. tetrasperma (Dodge, 1935; Mishra, 1971). However, the mating-type genes of these two species are capable of inducing mating-type incompatibility when introgressed into N. crassa background (Metzenberg & Ahlgren, 1973; Perkins, 1977). In the case for AT. tetrasperma, the suppression of mating-type incompatibility is due to the presence of the inactive tol locus (Jacobson, 1992). When tof is introgressed from N. T  crassa into N. tetrasperma, the heterokaryotic nature of that species breaks down upon germination (Jacobson, 1992; see chapter 5). It is possible that the absence of Tol activity is +  also responsible for the compatibility in other heterothallic and homothallic species. It is important to determine if tol is present in these species and what activity it might have.  4.2 MATERIALS  AND METHODS  4.2.1 Strains, media and culturing methods The N. crassa strains used in the study are listed in Table 4-1. Construction of strains R527 and R5-28 was described in section 3.2.2. The A/a partial diploid strain (D25) was constructed by selecting dual-mating (A and a) progeny in the cross R5-27 X R5-28. Culturing and crossing, using Vogel's (Vogel, 1964) and Westergaard's (Westergaard & Mitchell, 1947) media respectively, were performed as previously described, with modification (Davis & Deserres, 1970; Perkins, 1986). Mating tests were performed using/Z a (FGSC 4347) and/7 A (FGSC 4317) tester strains (Perkins et al., 1989).  For perithecial RNA extraction, crosses were performed in petri dishes containing Westergaard medium layered with Miracloth membrane (Calbiochem, La Jolla, CA). Perithecia were scraped from the Miracloth and RNA was extracted as described below.  110  Strain  Genotype  Origin  R4-71  his-5 tol trp-4; pan-2 a  This study  R4-72  ad-3B; trp-4 cot-1; pan-2 A  This study  his-5 A  F G S C 456  A2  ade-3A nic-2 cyh-1 a  A . J . F . Griffiths  1-20-41  ade-3B arg-1; tol A  A . J . F . Griffiths  1-20-26  ade-3B arg-1 A  A . J . F . Griffiths  1-10-1  ade-3A nic-2; tol a  A . J . F . Griffiths  74-OR23-1VA  Oak Ridge A  F G S C 2489  1-1-51  ade-3A nic-2 a  A . J . F . Griffiths  0R8-la  Oak Ridge a  F G S C 988  RLM44-02  thi-4; lys-1 A mat A  F G S C 8292  R5-28  T(IL->IIR)39311 ser-3A  This study  (Mauriceville background on L G I V ) R5-27  un-3; his-5 tol trp-4 a  This study  D25  his-5 tol A/a partial duplication  This study  R2-11  nic-2 al-1; pan-1 a"  N . L . G . Glass  fla  F G S C 4347  flA  F G S C 4317  P405  N. intermedia a  F G S C 1940  Kirbyville 8127  N. discreta a  F G S C 4378  SK-1 a  N. sitophila a  F G S C 5941  85A  N. tetrasperma A  F G S C 1270  W F S 5000  N. terricola (homothallic)  F G S C 1889  PR300  N. dogei (homothallic)  F G S C 1692  G349  N. galapagosensis (homothallic)  F G S C 4628  A-236  N. linolata (homothallic)  F G S C 1910  T R T C 51327  N. pannonica (homothallic)  F G S C 7221  N200  N. africana (homothallic)  F G S C 1740  S  133  Table 4-1. List of Neurospora crassa strains used i n this study. A l l strains are N. crassa Oak Ridge compatible (unless otherwise stated) and all tol alleles are N 8 3 . F G S C = Fungal Genetic Stock Center.  Ill  4.2.2 tol plasmid construction and transformation assay The 6.9 kb tol fragment was subcloned into pCB1004 (a hygromycin resistant vector; Carroll et al., 1994) from cosmids X 2 5 : D 7 , which contains T o l activity. C-terminal, N +  terminal, and internal deletion constructs of tol were made by using various restriction endonucleases. A frameshift mutation of tol was constructed by cutting the 6.9-kb tol/pCB plasmid with Xhol restriction endonuclease. The linearized plasmid (with the Xhol overhang) was then end-filled by Klenow fragment of Escherichia coli D N A polymerase I and religated. The result is a +1 frameshift mutation in tol after amino-acid position 50; the frameshift mutation was verified by D N A sequencing. E. coli DH5cc (Hanahan, 1983) was used as bacterial host strain for all plasmid amplification.  Spheroplast preparation and transformation were performed as previously described (Schweizer et al., 1981; Vollmer & Yanofsky, 1986) with the exception of selecting for resistance to hygromycin B (250 units/ml; Calbiochem). Constructs of tol were assayed for T o l  +  activity by co-transforming the cosmids (conferring hygromycin resistance) with a matA-1containing p O K E 1 0 3 construct (pOKE103 has a pan-2 selectable marker; J. Grotelueschen and R . L . Metzenberg, unpublished results) into strain R4-71 (his-5 tol trp-4; pan-2 a) and strain R 4 72 (ade-3B; trp-4 cot-1; pan-2 A) spheroplasts (see section 3.2.2).  4.2.3 Isolation ofto/-RIPmutants In N. crassa, duplicated sequences undergo methylation and GC—>AT mutation during mitotic divisions that precede karyogamy (Selker, 1990). T o order to obtain tol mutants, an internal tol copy (2.7 kb EcoRl fragment; D N A position 3-2735) was transformed into strain A 2 (ad-3A nic-2 cyh-1 a) and the transformant was crossed with a his-5 A strain. Since N. crassa transformants are heterokaryotic and contain both transformed and untransformed nuclei, a homokaryotic strain (4-9) with only transformed nuclei was isolated according to Ebbole & Sachs (1990). his-5 progeny (which contain the potentially mutated tol locus from parent 4-9) +  112  were isolated and assayed for T o l activity i n a heterokaryon test. For heterokaryon tests, 2 p i +  conidial suspensions (10 /ml) of two strains containing different auxotrophic markers were co7  inoculated onto vegetative growth media (Vogel, 1964). A compatible heterokaryon forms a vigorously conidiating culture after 3 days of incubation with a mean growth rate of 7 cm/day. Mating-type incompatible heterokaryons are usually aconidial and have a growth rate of approximately 0.7 cm/day (Vellani et al. 1994). Progeny without T o l activity were subjected to +  southern and R F L P analysis. The presence of altered restriction patterns in the R I P progeny is indicative of the GC—>AT mutations that occur during the R I P process.  4.2.4 Nucleic acid isolation/hybridization and Reverse Transcriptase PCR analysis Standard molecular biology procedures were used throughout (Sambrook et al., 1989). Genomic D N A isolation from Neurospora was adapted from Oakley et al, (1987). Total R N A was extracted according to Logemann et al. (1987) and enriched for poly(A)+ using the OligoTex m R N A kit (Qiagen, Chatsworth, C A ) . G e l electrophoresis and nucleic acids transfer to N y l o n filters (Schleicher and Schuell, Keene, N H ) and Zeta membranes (Bio-Rad, Hercules, C A ) were performed according to manufacturer's specifications. [ a - P ] d C T P (Amersham, 32  Oakville, O N ) labeled probes were generated from digested D N A using the T7 QuickPrime K i t (Pharmacia, Baie d'Urfe, P Q ) . For the hybridization experiment on various Neurospora species, 3 pg of each D N A was digested with EcoRl restriction enzymes before they were loaded into a 0.7 % agarose gel. For reverse transcriptase-PCR ( R T - P C R ) , c D N A s were synthesized by the Not I-d(T)6 primer using the procedure from First Strand c D N A Synthesis K i t (Pharmacia). P C R of D N A and c D N A were performed using the tol 13 (5' G G G C G G A G G A T A G G A G G 3'; bases 641 to 657) and tol 11 (5' C C A G C A G T G G C T C A G C 3'; bases 1144 to 1129) primers.  113  4.2.5 Yeast 2-hybrid system and screening of a cDNA/pAD-GAL4 library A 2-hybrid library for N. crassa was obtained from the Fungal Genetic Stock Center (FGSC) as aliquots of a phage suspension. It was constructed from mycelial cDNA (from conidia of FGSC 2489 grown in IX Vogel's and 2% sucrose for 24 hours at 25°C) unidirectionally ligated into the HybriZAP-2.1 vector (Stratagene, La Jolla, CA). Preparation of bacterial host strains (XLl-Blue MRF' and lambda resistant XLOLR) and fl helper phage (ExAssist) as well as amplification, titer determination and in vivo excision of the pADGAL4/mycelial cDNA phagemid (from the hybridZAP vector) were performed according to the instructional manual from the "HybridZAP-2.1 Two Hybrid Predigested Vector Kit" (hereafter referred to as the HybridZap System) (Stratagene, La Jolla, CA). The titer of the phagemid and helper phage were determined to be 3.5 x 10 and 3 x 10" pfu/ml, respectively. Mass library in 8  vivo excision of phagemid was performed using 4 x 10 XLl-Blue MRF' cells, 3.5 x 10 pfu of 9  8  amplified library phagemid (which should give at least 100 fold coverage of N. crassa primary transcripts) and 3.5 x 10 pfu of the helper phage. 9  The DNA binding domain vector used as bait in the 2-hybrid library screen was named tol/pGBT9. It is a fusion protein between TOL cDNA and the GAL4 DNA binding domain. A partial tol cDNA was obtained from an RT-PCR reaction using primers tol ATG1 (5'CCTGGGCTCACCTATGC 3'; base -50 to -34) and tol 11 (5' CCAGCAGTGGCTCAGC 3'; bases 1144 to 1129) from FGSC 2489 mRNA; the primer set brackets the only intron found in the tol ORF. After cloning into a PCRII vector using the TA cloning kit (Invitrogen, San Diego, CA), the partial cDNA fragment was swapped with its genomic counterpart from the vector tol/pGEM in order to create the full-length tol cDNA construct (named tol cDNA/pGEM). The complete tol cDNA (a Nsil-Nsil fragemt) was then introduced into the PstI site of pGBT9 (Clontech, Palo Alto, CA) to create an in-frame fusion TOL/GAL4-BD construct (named to*l/pGBT9). The in-frame fusion of tol/pGBT9 was confirmed by DNA sequencing.  114  The pAD-GAL4/mycelial cDNA target plasmid library and the tol/pGBT9 bait plasmid were transformed into yeast and were screened for protein-protein interactions between bait and target proteins by transcriptional activation of reporter genes. The reporter genes in yeast in this case were his3 and lacZ, and therefore the positive interactors should give a His and blue color +  colony. The yeast YRG-2 strain (Mata ura3-52 his3-200 ade2-101 lys2-801 trpl-901 leu2-3 112 gal4-542 gal80-538 LYS::UAS  GAL1  -TATA  GAL1  -fflS3 URA3::UAS  GAM  17m  ers x3)-TATA (  CYC1  -  lacZ) was used as host for transformation. Transformation of bait and target plasmids into yeast was performed according to the manual from the "YRG-2 Yeast Competent Cell Kits" (Stratagene, La Jolla, CA); 10 pg of each plasmid was used to transform 1 ml of yeast competent cells (with an efficiency of 4 x 10 colonies/ml). The transformed yeast cells were plated in solid 5  SD (synthetic minimal) media (1 ml per 150-mm plate) containing supplements and 5 mM 3AT (3-aminotriazole, a histidine antimetabolite) in order to select for positives with strong interactions. Cells grown in 3AT SD media without supplement of histidine were replicated, and were tested for LacZ activity in a filter lift blue/white assay (according to procedure from +  HybridZap System); their tolerance for different concentration of 3AT (from 5 mM to 25 mM) was also tested.  Isolation of plasmid DNA from yeast was performed according to the HybridZap System manual. The presence and size of inserts in pAD-GAL4 plasmid in the 2-hybrid positives were determined by PCR (according to protocol from the "GAL4 Vector Recombinant Screening Kit"; Stratagene) using the 5'AD (5' AGGGATGTTTAATACCACTAC 3'; nt 745-765) and 3'AD (5' GCACAGTTGAAGTGAACTTGC 3'; nt 929-949) primers. Partial sequence of the insert was determined by one run of DNA sequencing from the 5' end of the gel-purified PCR products. The pAD-GAL4 plasmids were transformed into MAX Efficiency DH5cc competent cells (Life Technologies, Rockville, MD) according to manufacturer's specification. The recovered plasmids were retransformed into yeast and the transformants were subject to the filter lift  115  blue/white assay for verification of interactions. Positive and negative controls for the assay were used according to the HybridZap System manual.  4.2.6 DNA sequence analyses D N A sequences were determined using the A B I automated Taq DyeDeoxy Terminator cycle method (Mississauga, O N ) at the N A P S unit, Biotechnology Laboratory, University of British Columbia. Computer sequence analyses for protein and D N A were done using the M a c V e c t o r / A s s e m b l y L I G N software (International Biotechnologies, N e w Haven, C T ) and G C G package available from the Wisconsin Genetics Computer Group (Devereux et al, 1984).  4.3 RESULTS 4.3.1 Functional analysis of tol constructs Several tol deletion and frameshift constructs were obtained to determine what portions of T O L are important for function (Figure 4-1). These constructs were subcloned into a hygromycin-resistant vector and were assayed for T o l activity. In the transformation assay, +  constructs of tol were co-transformed with mat A-l into a tol a strain and an A strain. If a construct retained T o l activity, it should trigger mating-type incompatibility in the tol a recipient +  and reduce transformation frequencies.  The results from the co-transformation experiments showed that a +1 frame-shift mutation at the Xhol site (aa position 50) abolished T o l activity, confirming that the 1011-aa +  O R E encoded T O L . Deletion of the portion including L R R and/or the S E T domain (a region of homology between H E T - S I X , H E T - E and T O L ) also abolished T o l activity. However, an +  internal deletion of 139 amino acids in the region before the L R R did not affect T O L function. N - terminal deletion constructs indicated that a region between aa position 98 and 531, which includes the coiled-coil domain and part of the S E T domain, was also essential.  116  1936  -2600 EcoRI  Sma\  \  148 .Q^Xhd 290  -818  Sa/I  Spei  2041 2458 NaA Nai\  1688 Cla\  H/'ndlll  2491 ^A/ael 3400 V NsK  4300 EcoR\  6.9 kb tot* tol  B  TOL Ecx)R\-EcoR\ Sal\-EcoR\ Spe l-A/s/1  Coiled-coil  SET  177-211  315-583  tol+ activity  LRR  T  804-823  1011 aa  +  Spe \-Nae\ 801  EcoR\-Sma\ 615  i  EcoR\-Cla\  r  533  Xho\ frameshift 50*  I  Cla\-EcoR\ 532  Hind\\\-EcoR\  2_  98*  A Nah-Nati 1  i  r  651  791  1011 1011  1011  Figure 4-1. Functional analysis of tol deletion and frame-shift constructs. ( A ) Schematic representation of the 6.9 kb tol D N A sequence including relevant restriction sites. D N A position is given according to Figure 3-3. (B) Schematic diagram of various tol constructs used to examine functional domains of T O L . The three putative functional motifs in T O L are the coiledcoil domain, the L R R (leucine-rich repeat) and the S E T domain (a region o f homology between H E T - S I X , H E T - E , and T O L ; see Figure 3-5). A m i n o acid position is according to Figure 3-3. A tol construct is considered to have T o l activity i f it results in a reduction i n transformation +  frequency (at least 20-fold) when co-transformed with mat A-l into tol a spheroplasts, as compared to frequencies when co-transformed into A spheroplasts. *XhoI frameshift contains 50 functional amino acids and terminates at position 68. ** The Hindlll-EcoRI  construct could use  a vector A T G as start codon. O n the other hand, it could use an internal alternative in-frame A T G site (at amino-acid position M174) to initiate translation. Alternative in-frame initiation of translation is also inferred to occur with het-6 (Smith et al, 2000b). 117  4.3.2 Isolation ofto/-RIPmutants The mutation in the original tol' allele (N83) occurred at the 3' end o f the gene (near the carboxyl-terminus of T O L ) , and therefore it is possible that T O L retains partial function. Attempts were made to obtain additional tol mutants by repeat induced point (RIP) mutation (Selker, 1990). A 2.7 kb EcoRV fragment (cloned into pCB1004) that contained an internal portion of tol (position 3 to 2735, Figure 3-3) was transformed into strain A 2 (ad-3A nic-2 cyh-1 a; Table 4-1). A hygromycin-resistant homokaryotic transformant (#4-9) was obtained and crossed with F G S C 456 (his-5 A). Forty-nine his-5 ade-3A nic-2 a progeny were analyzed for +  Tol  +  activity in heterokaryon tests. Five his-5 ade-3A nic-2 a progeny formed vigorous +  heterokaryons with the tol A tester (1-20-41), indicating that the tol allele in these strains was not functioning to confer mating type associated incompatibility (Figure 4-2A). Heterokaryons between these same five his-5 ad-3A nic-2 a progeny and the tot A tester (1-20-26) displayed +  typical heterokaryon incompatibility (Figure 4-2A), indicating that the tol mutation in these strains was recessive. The to/-RIP strains were phenotypically normal during vegetative growth and mated as either a male or a female, with similar fertility to their parental strains. Thus, these five progeny displayed a phenotype identical to the original tol mutant and were thus designated as to/-RIP strains (tol-43, tol-83, tol-95, tol-106 and tol-135).  To confirm that the suppressor phenotype in the tol-RIP mutants segregated with the tol locus, tol-43 a (as a male) and tol-83 a (as a female) were crossed separately to his-5 A. O f the 30 H i s progeny tested from the 2 crosses, all but one formed vigorous heterokaryons with both +  tol a (1-10-1) and tol A (1-20-41) strains (3.3% recombination), indicating that the new mutations in tol-43 and tol-83 were closely linked to the his-5 (and hence tol) locus. B y Southern blot and R F L P analysis, it was determined that the tol sequence in the tol-RIP strains showed restriction site changes (Figure 4-2B), consistent with the presence of transition mutations that are characteristic of sequences that have undergone R I P (Selker, 1990).  118  A tol A +tol a  A* tol a  A +to^RIPa  A+ a  tol A+fcri-RIPa  B tol-43 tol-83 tol-95 tol-106 tol-135 M  S  M  S  M  S  M  S  M  S  M  S  A2 hls-5  #4-9 M  S  M  S  Figure 4-2. Phenotypic and genotypic analysis of to/-RIP mutants. ( A ) The top three plates are controls showing growth characteristics of compatible (tol A + tol a) and incompatible (A + tol a and A + a) heterokaryons. The bottom plates show the phenotype of R I P strain tol-43. The tol43 strain was compatible with a tol A strain, but incompatible with an A strain. Strains 1-20-41 (tol A), 1-20-26 (A), 1-10-1 (tol a), 1-1-51 (a) and R5-52 (tol-43 a) (Table 4-1) were used in heterokaryon tests. (B) R F L P and cytosine methylation analysis of tol sequences in the to/-RIP strains. Genomic D N A from parental strains (his-5 and transformant #4-9) plus the transformation recipient strain ( A 2 ; Table 4-1) and each to/-RIP strain was digested with Mbol ( M ; cytosine methylation-insensitive) and Sau3AI  (S; cytosine methylation-sensitive) and probed  with a 3.5 kb fragment containing tol. The presence of altered restriction patterns in the R I P progeny as compared to their parental controls is indicative of GC—>AT transition mutations that occur during the R I P process (Selker, 1990). 119  4.3.3 Expression analysis of tol by RT-PCR The exact role of tol in mediation of mating-type-associated incompatibility is unclear. One possibility is that tol is a downstream effector of incompatibility whose expression is regulated by the mating-type proteins. Transcripts of tol were not detectable by R N A hybridization analysis (P.K.T. Shiu, unpublished results); therefore the expression of tol was analyzed by reverse transcriptase (RT)-PCR. Primers bracketing the intron (tol 11 and tol 13) were used to amplify tol m R N A from growing vegetative cultures. Figure 4-3 shows that a tol c D N A could be detected in the A, a and a mating-type deletion strains grown in vegetative (V) and crossing (C) media (V and C ; Figure 4-3). These data indicated that neither mating type constitution nor growth conditions materially affected the expression of tol. A tol c D N A could also be detected in an Ala tol partial diploid strain, indicating that the presence of both mat A and mat a in the same nucleus did not significantly alter activation or represssion of tol. Expression of tol was also detectable in both an incompatible (A + a inc.) and compatible heterokaryon (A + a ); the a m33  m33  strain forms compatible heterokaryons with A strains, but sexual function is not  affected (Griffiths & Delange, 1978).  Following fertilization, opposite mating type nuclei co-exist and divide within a common cytoplasm. Therefore, mating-type associated incompatibility mediated by tol must be suppressed during sexual reproduction. T o determine i f tol is transcriptionally repressed during a cross, thus allowing the co-existence of opposite mating type nuclei, the presence of the tol c D N A was analyzed by R T - P C R in m R N A prepared from perithecia at 3, 5, 6, and 9 days postfertilization. Although a c D N A for mat A-2 could easily be detected by R T - P C R from perithecial R N A preparations for all time points, a tol c D N A could not be detected at any postfertilization time point (cross; Figure 4-3).  120  A/a A  a  Lmat  tol  A  a  Amat  A/a  M A+a a™  tot cross  inc. com. DNA  33  Figure 4-3. Expression of tol as analyzed by R T - P C R . Primers bracketing the intron (tol 11 and tol 13) were used to amplify tol c D N A from different m R N A preparations. The size of the genomic tol product is 504 bp with these primers, whereas the tol c D N A product is 410 bp. Strains used are as follow: 7 4 - O R 2 3 - 1 V A (A), O R 8 - l a (a), R L M 4 4 - 0 2 (A matA), D 2 5 (A/a tol), I-20-26/I-1-51 (A+a) and I-20-26/R2-11 (A+a™ ) tol m R M A was isolated from the above 33  cultures grown either in vegetative medium (Vogels, 1964; V) or crossing medium (Westergaard and Mitchell 1947;  C) or from  perithecia  (cross).  A tol c D N A could be detected in m R N A from  all strains, with the exception of from perithecial m R N A . The quality of the perithecial m R N A samples was checked by the amplification of a mat A-2 c D N A (using primers r l . l and 24232406; Ferreira et al., 1996); mat A-2 is expressed throughout sexual development (data not shown). D N A size marker (1 kb ladder) is given on the left.  121  4.3.4 Phenotypic studies of tot mutant as complemented by different tol constructs Efforts were made to study the sexual and vegetative phenotypes of Tol transformants +  under native and constitutive promoters.  Five different constructs of tol (with different  promoters) were made and they were subcloned into a Pan-2 vector (pOKE103) (Table 4-2). +  They are tol6.9 (with the 2.6 kb native promoter), tol5.1 (with the 0.8 kb native promoter), tol4.3 (with the 63 bp native promoter), ptrpC-tol (with the trpC promoter fused to tol), and pcrp2-tol (with the crp-2 promoter fused to tot). The trpC promoter is a constitutive promoter from the A. nidulans trpC gene (Mullaney etal., 1985) whereas the crp-2 is a constitutive promoter from the N. crassa ribosomal gene (Cujec & Tyler, 1996). A tot pan-2' strain (R4-71) was transformed with the above five constructs and several homokaryotic transformants were obtained. The Pan2 homokaryotic transformants were then tested for Tol activity in a complementation test. +  +  As expected, homokaryotic transformants conferring Tol phenotype can be obtained +  from each of the five transformation experiments; these transformants failed to form vigorous heterokaryons with the opposite mating-type, showing that they can complement the original tot mutation, ectopically. They do not have a vegetative phenotype different from that of a wildtype strain. When used as males in a cross, fertility was unaffected; the number of perithecia formed, the time required for perithecia development and the maturation of spores was normal. However, Northern analysis (during vegetative growth) demonstrated that Tof transformants from ptrpC-tol- and pcrp2-tol-mediated transformation (D and I series; Table 4-2) do not contain over-expressing tol transcripts (data not shown). The failure in obtaining over-expressing tol constructs can be due a phenomenon called quelling (Cogoni et al, 1996). In N. crassa, a transgene-induced silencing mechanism occurs that represses (reduces) expression of a transgene. It is thought to be mediated by RNA-RNA interaction between resident gene and transgene (Cogoni & Macino, 1999). Alternatively, the transgenic tol copy could be introduced into an ectopic site that does not allow the proper transcriptional control.  122  Tol transformants  Constructs Promoter region  tol6.9  +  -2600  tol -818  tol5.1  E4, 13, 27,43  JQL  F4, 5, 15,42  JQL  G18, 38  ptrpC-tol  JQL  D10, 17, 19, 28, 35  pcrp2-tol  JQL  11,4, 12, 24, 27  -63,  tol4.3  Table 4-2. Constructs used in complementation of tol mutant. Five constructs of tol, under native and constitutive promoters, are used in complementation experiment. tol6.9 (EcoRlEcoRI fragment; see Figure 4-1), tol5.1 (Sali-EcoRl), and tol4.3 (Spel-Nsil) are constructs of tol gene under native promoters of 2.6 Kb, 818 bp and 63 bp, respectively. ptrpC-tol was constructed by fusing the promoter region (-357 to -2) of the trpC gene from A. nidulans (Mullaney et al, 1985) to construct tol4.3. pcrp2-tol was constructed by fusing the promoter region (-438 to -1) of the crp-2 gene from N. crassa (Cujec & Tyler, 1996) to construction tol4.3. The five constructions were then transformed into a tol' strain (R4-71). Homokaryotic transformants conferring Tol phenotype can be obtained for each transformation and they were +  listed on therightmostcolumn. The Tol transformants were then tested for their sexual +  phenotype as males. Results showed that none of the transformants are affected in their fertility.  123  4.3.5 Detection of tol sequences in other Neurospora species A 3.5 kb Spel-Nsil fragment (position -63 to 3407, Figure 3-3) was used as a probe in Southern analysis using genomic D N A from N. crassa (both the wild type and the tol mutant) and ten other Neurospora species. Three u.g of D N A was loaded into each lane and quality and quantity of genomic D N A were confirmed by hybridizing the blot with a (3-tubulin gene probe (Orbach et al., 1986). Under high stringency of hybridization (60°C), only one copy of tol was detected in N. crassa (Figure 4-4); although sequences that encode T O L - l i k e gene product are present in N. crassa genome, they do not have significant D N A sequence similarity with tol (see section 3.3.4). The tol mutant has the same size band as the wild type, confirming that the tol' phenotype is due to a point mutation, rather than due to a large D N A deletion as observed in het6 escape events (Smith et al, 1996).  Sequences that hybridized to tol were also detected under high stringency in many different Neurospora species, including all the heterothallic and pseudohomothallic species, and four of the six homothallic species tested (Figure 4-4). These data show that tol may be e v o l u t i o n a r y conserved and present among other Neurospora species. N. tetrasperma, the only other species listed in Figure 4-4 with mating-type-associated incompatibility, has a similar size band as N. crassa. Interestingly, in D N A from homothallic strains which contain sequences of both mat A and mat a (i.e. N. terricola and N. pannonica), foMike sequences cannot be detected. It has been shown that mat A-l from N. pannonica confers mating-type-associated incompatibility in N. crassa and that mating-type compatibility in N. pannonica is probably due to a toMike suppression (see section 2.3.5).  124  23 Kb 12 Kb 7 Kb  1.6 Kb Figure 4-4. Detection of tol sequences in various Neurospora species. Genomic D N A was digested with EcoRl and probed with the tol 3.5-kb Spel-Nsil fragment. Species names and the presence of mating-type sequences are given (A+a = contains both mat nuclei; A/a = homokaryon with both mat genes) (Pbggeler, 1999). A m o n g the Neurospora species listed here, N. crassa and N. intermedia are the only species that confer mating-typeassociated incompatibility. N. sitophila and N. tetrasperma are mating-type compatible species with a foMike suppression (Perkins, 1977; Jacobson, 1992). The crassa strains used are #43 (tol) and 7 4 - O R 2 3 - I V A (wild type). Other species used are listed in Table 4-1.  125  4.3.6 Isolation of top (TOL-interacting protein) genes Efforts were made to isolate TOL-interacting proteins using yeast 2-hybrid system. The yeast 2-hybrid system is used to detect protein-protein interaction in vivo (Fields & Song, 1989). When a protein X, which is fused with the GAL4 DNA binding domain, interacts with a protein Y, which is fused with the GAL4 activation domain, the GAL4 transcriptional activator is reconstituted and transcription of reporter genes are therefore activated.  A complete tol cDNA sequence was constructed using RT-PCR and was fused with the DNA binding domain of the yeast GAL4 gene (see Materials and Methods). This fusion construct, tol/pGBT9, was used in screening a N. crassa mycelial cDNA library (fused with GAL4 activation domain). The reporter genes in yeast in this case are his3 and lacZ, so the positives should give a His and blue color colony. +  Thirty positives were isolated from the 2-hybrid screening (Table 4-3). Out of the 30 positives, only 5 were shown to be in-frame with GAL4 activation domain and have plasmiddependent interaction with TOL. Four cDNAs encode novel genes (clones 7A5, 7A10, 7D3 and 7C10). The most interesting positive is clone 7D8, which is similar to vipl (p = 9e-39) from fission yeast (Figure 4-5). vipl was isolated from a 5. pombe expression library using human p53 antibody (P. Wagner, unpublished results). P53 is a cancer suppressor in humans and is involved in cell cycle regulation and apoptosis (Sheikh & Fornace, 2000). A vipl homolog is apparently present in other Neurospora species (including species that do not mediate matingtype-associated incompatibility) (see Appendix 8.4)  126  c  4) co ^  4j  3 2  4>  03 - O O C  al  4> 00  co  c  fl 4) fl 3  <loo  CM  CN  A  CN  DH  ON  NO  to |oo  ato NO NO CO o o 2 o <o  2 5 O  ll  a  I  c o 3  o s o  ato to 2 s fl -fl 03  s to  c H  W  ^ ,  a  In  'I  o u  ID co co 03  4>  o  S i>  fl .o  -fl o >  &  o > o  ll  —H I  03  S O  I  4)  |£ c o  "aT o fl o3  c  u. M|  « co  M  z, Q  c o "3 c o  I  Ml  CO  s  a  00  co 1) *-i .  2  OH  4)  fl  4  u  CO  fl  co  u  O 3 Ml  o  Sd  4)  s CO  .23 «  CO  IS  a  <u O O 3  M J  C  « fn  4) O  C  CO  4) co O O 3 Ml  OH  O  to  c  8,  CO  a o  |T3 4) co  s,  CO  2  s ,  Vi  0.-A  I  T3 <U co  03 Mfl  CO  i-H  fl  g  S a  as i-o 3  «—»  X o  s  .  to .  3 to O 3 <u, <D OH OH fl . C4) CO co > O-J W)| t>0| O 3 O C im l-fl C c bf)l o3 fl o  2  <  O  co  o  A  Onf  4> 3 '-fl  »3  f  e o o  o  s  I*  -fl o  >  CO  t-i  ON  4>. 60  2 s  1)  Sc a s &  4) to £ 4) M N  •  2 c  to  a  2  ra to • -^ j-i M T ! 4)  4>  ON ON ON  ll  Z 3 ^ c n e 4) > ^  o  OJ  5 K .gS <a<op q  sill  13  ^-S  (D C .5  3  CO  oj  O 3 *^ c  M  O _§ 4)  03  <u c  a  o  4) co 00 4) ' 4) .OH  oo o o o o o o o o © o O I© o O l© o o o I© o o I© o o © ooo in © o o oo o o o CN CO o o CN in o © o o © in o in in in N O NO ON ON «n O N in in  e o U  oo  CN  iOO  OJX)  >  |T3 ^ „  O in  ° S fl  ON  ^  <ui  !U  m  <n  CO  V S  g  OH  4)  I  4>  > ra  M  U  M  co C ,O O,  co  .  e 4 > .-fl  3^V o o £ & fl  3  &, 4) *  127  7D8 VIP1  10 20 30 40 50 MST-VYVKNIGANTEEKDIRAFFSFCGKISSLDVTTEGETKSATVTFEKESAARTALLLD MSNQVIVTNISPEVTEKQISDFFSFCGKVSNISTEKSGETQTAKIQFERPSATKTALLLQ 10 20 30 40 50 60 60  7D8 VIPl  7D8  70 80 90 100 110 HTKLGEHELSVTSASGEHADSGDNVHPKSDADRDTDEITQEEKPRARVLAEYLASGYLVA  DALLGQNKIQITSEDGGAASTTD 70 80 120 130 DSGLKTAIALDEKHGVSQRF  QGGAGGD-QAARQEDKPRSAIISELLSRGYHLS 90 100 110 140 150 160 170 L S TIQNL DQKYHATDRAKTADQ S YG ITARANS L  VIPl  DVTLEKSIQLDQSYGVSSKFKGILESALSGVRSVNERYHVTEKANEVDNKFAISDKLNRT 120 130 140 150 160 170  7D8  FSGLSSYFEKALE-APGA  VIPl  SSLVSTYFHKALETAAGTSAGQKVQNAYEDGKNQLLGIHNEARRLADAKNQAEGTAS 180 190 200 210 220 230  180  7D8 VIPl  190  200  210  220  KKIVDFYTTGSKQVQDIHNEAKRLAELKKQEAGGSSYKA  230 240 250 260 270 280 AGLDKIFGAEKAPGQESKPNDQVPGAAPSDAAATESNQQPISEGAYPGTAEKIPQ PAS STP - TAPAE  KE PTA  240  PTTESKTTE  250  Figure 4-5. Similarity between the predicted amino-acid sequences from a top gene (7D8) and VIPl. Top strand: amino-acid sequence for 7D8 (283-aa). Bottom strand: amino-acid sequence for VIPl (257-aa). vipl is a p53-related gene isolated from S. pombe. The two proteins have 33.9% identity and a p-value of 9 x 10" for similarity. ":" denotes identity whereas "." denotes 39  conservative change of amino-acid (Devereux et al., 1984).  128  4.4  DISCUSSION This chapter describes the characterization and expression pattern of tol, a mediator of  mating-type associated incompatibility in N. crassa, as well as the isolation of additional tol mutants and potential genes (top) that encode TOL-interacting proteins. Functional analysis of various deletion and frameshift constructions confirm that the tol ORF identified indeed confers Tol function as tested in a transformation reduction assay. Furthermore, the functional analysis +  identified regions of TOL that are important and regions of TOL that are dispensable for proper function. For example, regions from amino-acid 1 to 97 and from amino-acid 652 to 790 are nonessential for Tol function while regions from amino-acid 802-1011 and from amino-acid 99 +  to 521 are absolutely required. The required regions include the coiled-coil domain and the LRR motif. It is possible that the two protein-protein interaction domains are essential for the Tol  +  activity. If this is the case, then TOL probably mediates mating-type incompatibility through protein-protein interactions.  Since the original tol' allele (N83) may retain partial activity, five additional tol mutants were isolated through RIP (repeat-induced point) mutagenesis (Selker, 1990; Singer & Selker, 1995). The fo/-RIP mutants isolated (tol-43, tol-83, tol-95, tol-106, and tol-135) behave like the original tol mutant - they were phenotypically normal during vegetative phase and can mate as either a male or a female, with similar fertility to wild-type strains. On the other hand, mutations in mod genes, the mediators of non-allelic incompatibility in P. anserina, yield phenotypes (especially during sexual development) other than vegetative compatibility (see section 1.4.8). The mod-Al mod-Bl double mutant, which suppresses growth and lytic defect in selfincompatible (SI) strains, has a defect in protoperithecia formation and is female sterile (Barreau et al., 1998). Mutations in the mod-D gene (Loubradou et al, 1999), which mediate lytic action in certain incompatible strains, have defects in aerial hyphal growth, protoperithecial formation, spore germination and secondary ramifications (Durrens et al., 1979; Labarere & Bernet, 1979a,  129  b). Mutation in the mod E gene is responsible for both partial restoration of the growth of a SI het-R het-V strain and a sexual dysfunction (less asci with mature spores and higher frequency of uninucleate spores) (Loubradou et al., 1997). It has been proposed that het and mod genes were involved in perithecial formation (Bernet, 1992). In order to direct all growing potential toward the formation of perithecia, growth arrest and partial lysis of hyphal cells surrounding the developing perithecia is initiated through a pathway controlled by the het and mod genes. However, mutations in tol do not affect protoperithecial or sexual development.  In N. crassa, it is not clear how tol, mat a-1 and matA-1 function to trigger incompatibility during vegetative growth. One hypothesis is that, in an A + a heterokaryon or A/a partial diploid, M A T a-1 and M A T A - l form a heterodimer and this complex acts as a transcriptional regulator to activate or repress the expression of tol (Glass & Staben, 1990), which would then trigger incompatibility. Our studies show that the expression of tol occurs in A, a and even Amat strains. These data indicate that M A T A - l and M A T a-1 are not required for either repression or activation of tol, although we cannot rule out the possibility that the matingtype polypeptides may modulate tol expression. The expression of tol was also detected in a A/a tol partial diploid and an A + a incompatible heterokaryon, indicating that if a M A T A - l / M A T a1 heterodimer is formed, it does not materially affect the expression of tol.  Based on the data presented in this study, a second more plausible possibility for the mechanism of mating-type associated incompatibility can be formulated. In our current hypothesis, TOL physically interacts with the M A T A - l / M A T a-1 complex or their downstream products to trigger incompatibility. The 1011-aa TOL protein possesses a putative coiled-coil domain and a LRR, both of which are thought to mediate protein-protein interactions. Putative LRRs are found in both M A T A - l and M A T a-1 (see Chapter 2) and alteration of these motifs affect the capacity of M A T A - l or M A T a-1 to induce incompatibility. In support for the model of a T O L / M A T A - l / M A T a-1 multimerization, physical interaction has been detected between 130  MAT A - l and TOL and between MAT a-1 and MAT A - l via the yeast two-hybrid system (C. Staben, T. Badgett & P.K.T. Shiu, unpublished data). A deletion within the first LRR of MAT a1 eliminates interaction with MAT A - l in the yeast two-hybrid system and abolishes mating-type incompatibility but does not affect mating function. For future experiments, interaction between TOL, MAT A - l and MAT a-1 should be confirmed by immuno-precipitation and various mutants that eliminate incompatibility (such as tol' and other mat A mutants) should be assessed for their ability to mediate protein-protein interaction.  As mentioned in chapter 3, the tol mutation has been shown to suppress the phenotype of a fmf-1 mutant (Johnson, 1979), which is sterile as both a male and a female in crosses to a wildtype partner. Fertility is restored when a fmf-1 tol strain is crossed to a fmf-V tol strain. Our expression analysis of tol is consistent with the hypothesis that tol activity is suppressed during sexual development to allow the co-existence of opposite mating-type nuclei in the ascogenous hyphae. It is possible that FMF-1 regulates tol either directly or indirectly, and thus may play a role in the repression of tol transcription; mutation in fmf-1 may allow expression of tol during sexual development and hence trigger mating-type-associated incompatibility. The presence of short repetitive sequences in the promoter of tol may provide putative binding sites for such regulators. Presumably, the mis-expression of tol during the sexual cycle would result in matingtype-associated incompatibility and block further sexual development. Attempts have been made to create constructs that over-express tol during sexual development. However, the constructions containing constitutive promoters (from crp-2 and trpC genes) do not yield over-expressing tol transcripts. It is possible that these promoters may not work as expected in ectopic locations in the transformants or that the distance between these promoters and tol ORF may not be optimal. Alternatively, there may be sequences in tol mRNA that target it for rapid degradation. Until a consistent constitutive or inducible expression system during sexual development is established in N. crassa, we cannot unambiguously determine the effect of over-expressing tol during sexual development. Inducible expression systems (e.g. ars-1 promoter; Paietta, 1989 and unpublished 131  results) and genes that are highly expressed in sexual tissue (e.g. poi-1 and poi-2; M.A. Nelson, unpublished results) have been described. Further development of these systems may provide useful expression tools during sexual development in N. crassa.  Sequences similarity with tol are detected in many different Neurospora species (Figure 4-5), including those from heterothallic (nonself-mating, single nuclei), pseudohomothallic (selfmating with opposite mating-type nuclei), and homothallic groups (self-mating, single nuclei). These data show that the tol gene may be present in some of those species. The tol locus apparently plays a crucial role in the life style of mixed mating-type pseudohomothallic and some homothallic species. For example, the inactive tof allele evidently allows co-existence of incompatible mating-type nuclei in a common cytoplasm in the pseudohomothallic species N. tetrasperma. When the tof gene is introgressed from N. crassa to N. tetrasperma, the otherwise heterokaryotic species broke down into homokaryons of opposite mating-type (Jacobson, 1992). In some homothallic species, like N. terricola and N. pannonica, both mating-types genes are present in the same nucleus and yet an incompatible reaction is not triggered. The fact that a tollike sequence is not detectable in those species may explain why mating-type incompatibility is not present (Figure 4-5). It is possible that the natural adaptation of tol may play an important role in the evolution of homothallic and pseudohomothallic species since mating-type incompatibility may present a problem in species that contain both mating-type genes. A to/-like suppression is also likely to be present in some heterothallic species. For example, mating-type incompatibility is observed in N. intermedia isolates, but not in N. sitophila or N. discreta isolates. When mating-type genes from N. sitophila were introgressed into N. crassa, it was found that these genes confer mating-type incompatibility and therefore suppression of incompatibility must be due to atof-likegene (Metzenberg & Ahlgren, 1973). The molecular characterization of tol in these species will provide great insight into the functions of tol as well as its relation with the evolution of reproductive life-styles in these species. It may provide  132  answers to interesting questions such as why tol-like sequences are retained in species that do not mediate mating-type-associated incompatibility.  To understand the role of T O L in mating-type heterokaryon incompatibility and to identify other proteins involved in the process, several potential TOL-interacting proteins were identified using the yeast 2-hybrid system. Five cDNAs encoding potential TOL-interacting protein (top) were identified, with four of them encode novel sequences. One potential top gene encodes a protein with similarity to the predicted products of the vipl gene from fission yeast. vipl was originally isolated from a S. pombe expression library using the human p53 antibody. P53 is a cancer suppressor in human and it is involved in cell cycle regulation and apoptosis (Sheikh & Fornace, 2000). Over-expression of a mutant form of vipl causes growth arrest in S. pombe (P. Wagner, personal communication). It is possible that the N. crassa vipl homolog is involved in cell cycle/cell death pathway downstream of antagonistic reactions between matingtype (and even other het loci) gene products. Interaction between V I P l and T O L may trigger the growth arrest pathway through signal transduction. If V I P l is indeed involved in the function of TOL, its function may not be limited in mediating mating-type incompatibility, since vip-1 sequences can be detected in Neurospora species that do not confer mating-type incompatibility. RIP (repeat-induced point) mutational analyses of these top genes are under way (M.D. Hiltz & N.L. Glass, unpublished data) and the phenotype of the mutants will reveal their functions, as well as their role in mediating mating-type-associated heterokaryon incompatibility.  133  5. Analysis of tof and its relation to non-allelic and sexual incompatibility 5.1  INTRODUCTION N. tetrasperma, a pseudohomothallic species, is a naturally occurring self-fertile A + a  heterokaryon; four rather than eight ascospores are produced in each ascus, and both opposite mating-type nuclei are packaged in the same ascospore (Sansome, 1946; Raju, 1992a). Because of its heterokaryotic life-style, the mating-type incompatibility must be suppressed in this species. The mating-type genes of N. tetrasperma are not responsible for the suppression of incompatibility because mat A and mat a from N. tetrasperma displayed mating-type incompatibility when introgressed into N. crassa (Metzenberg & Ahlgren, 1973). The suppression is apparently due to the absence of Tol activity. This can be shown by the +  introgression of tof (wild-type tol from N. crassa) into N. tetrasperma, which induced matingtype-associated incompatibility and disrupted the pseudohomothallic nature of the species. Upon germination, the opposite mating-type nuclei that were together in each ascospore disassociate, resulting in a mixed culture of segregated A and a mycelia instead of an A + a heterokaryon (Jacobson, 1992). Furthermore, the tof allele (wild-type tol from N. tetrasperma) acts as a recessive suppressor of mating-type incompatibility when introgressed from N. tetrasperma to N. crassa (Jacobson, 1992). The tof allele can therefore be considered a natural tol' mutant.  Complications occurred during the introgression of the tof allele into N. crassa and provided evidence that tof, or a locus closely linked to trp-4 and tof, may function to reduce fertility and to mediate non-allelic incompatibility with an unlinked locus (Jacobson, 1992). The complications encountered were the observation of unanticipated reduced fertility during the backcrosses (of tof from N. tetrasperma into N. crassa) and the occurrence of a class of progeny with a deleterious growth phenotype.  134  Complication 1: Reduced fertility was observed when the to? trp-4* progeny (from the to? to N. crassa introgression backcrosses: to?trp-4* xN. crassa tof trp-4) were crossed with N. crassa. A higher proportion of perithecia arrested early in development with delayed development of perithecia and ascospores (Figure 5-3B). The reduction in fertility occurs whether a to? or to? strain is used as the female parent. The tof trp-4' siblings do not show such phenotypes when backcrossed to a N. crassa strain.  Further characterization of the phenomenon provided evidence that the reduced fertility observed was mediated by a locus closely linked to tol and trp-4 (D.J. Jacobson, unpublished results). Interestingly, the sexual incompatibility can be suppressed by a mutation in mating-type gene mat a-l. The fertility pattern of the fo/ -introgressed strains can be summarized as follow: 7  mating-tvpe-associated Strain 1  Strain 2  vegetative incompatibilitv  Fertilitv  A; tof  a; tof  incompatible  Low  A; tof  a; tof  incompatible  Low  A; tof  a; tof  incompatible  Normal  A; tof  a; tof  compatible  Normal  A; tof  a \ tof  compatible  Normal  A; tof  a \ tof  compatible  Low  m33  m33  From the above data, it can be interpreted that tof, or a locus introduced with tof during its introgression to N. crassa (i.e. a locus closely linked to tof and trp-4) mediates the reduction in fertility. The lowered fertility is observed when the two mating partners have heterologous alleles (one from N. crassa and one from N. tetrasperma) at tol or at a closely linked locus. This fertility reduction can by suppressed by the a  m3S  allele, which has a R258S mutation in the mat a135  1 gene that suppresses mating-type incompatibility but does not affect mating function (Griffiths & DeLange, 1978; Staben & Yanofsky, 1990). However, the suppression is only observed when the a  m33  mutation is present in the same nucleus as the tof allele.  One hypothesis to explain the above observation is that the tof Itof combination somehow triggers heterokaryon incompatibility between alternative mating-types during fertilization; normally, mating-type incompatibility is suppressed during mating. The tof-tof interaction may act to nullify the suppression by an unknown mechanism. Since the a  m3S  allele is  unable to mediate incompatibility between opposite mating-types, its presence thus prevents the occurrence of incompatibility and allows normal fertility between a tof and a tof strain. This model, however, does not give any explanation (or contradiction) for the observation that a  m33  must be present with tof in the same nucleus for normal fertility to occur.  Although recombination has not been observed between tol and the sexual incompatibility locus, there is no direct proof that tol is responsible for the sexual incompatible phenotype. Therefore, the newly discovered sexual incompatibility locus will be referred to as the 1st (intergenus sexual incompatibility) locus, with isf and isf being the sexually incompatible alleles.  Complication 2: Although a cross between a tof andto/ -introgressedstrain shows reduced fertility (see r  above), a few progeny can still be recovered. In the ninth backcross of tof from N. tetrasperma into N. crassa (b tof trp-4 ; leu-3 axN. crassa tof trp-4; arg-1 A), inhibited progeny and +  8  skewed ratio of markers were recovered (Jacobson, 1992). Among the ascospore progeny, 1/4 show inhibited growth. The inhibited progeny class show phenotypes resembling the "dark agar" seen in strains with a partial duplication heterozygous for a het gene (Mylyk, 1975). In the viable ascospore progeny, all the tof progeny are heterokaryon compatible with their tof parent, 136  while the tof progeny are split into two groups: half are compatible with the tof parent and the other half are incompatible. One hypothesis to explain the presence o f a deleterious class and the skewed ratio is that a non-allelic incompatibility reaction is mediated by 2 unlinked loci (with one of the loci linked to tol). The observed mutually exclusive incompatibility group can be explained as the following, with the newly discovered incompatibility loci named ///-/ and ili-2 (inter-locus incompatibility):  Progeny Groups tof parental b9 progeny Genotypes  tof parental  ili-2  T  T  Inhibited  parental  progeny  35  36  34  37 ili-l  tof non-  ili-l  c  ili-l  ili-2  c  c  ili-2  T  ili-l  The deleterious class in this case contains the antagonistic ili-l  T  T  total 142  ili-2  c  (from ./V. tetrasperma)  and ili-2 (from N. crassa) alleles. The non-parental group in the progeny is ili-l c  c  incompatible with its tof parent (ili-l  ili-2 ). c  c  ili-2 , which is T  Non-allelic incompatibility has been well  documented in P. anserina (Bernet, 1965; Bernet et ah, 1973; for review, see chapter 1.4), but it has not been widely characterized in N. crassa. Recently, Smith et al (2000b) has proposed that the /ler-fj-mediated incompatibility in N. crassa, once thought to be mediated by a single locus, is mediated by two linked genes (see section 1.4.4).  ili-2 is closely linked to het-c c  0R  (no recombination is observed in 500+ progeny) and  pyr-4 (D.J. Jacobson, unpublished data), het-c is a heterokaryon incompatibility locus that mediates allelic incompatibility in N. crassa (Saupe et ai, 1996a; see Chapter 1.4 for review). On the other hand, ili-l  T  is closely linked to tof and trp-4. Recombination between ili-f  tof has not been detected in 400+ progeny; only two recombinants between ili-l  T  and  and trp-4 were  recovered (D.J. Jacobson, unpublished data). Judging from the linkage of the phenotypes, it is 137  possible that tof and het-c may be identical to the two Hi loci and that they could mediate nonc  allelic incompatibility.  The focus of this study is to determine (a) if a single locus in N. tetrasperma is responsible for the ili-l non-allelic incompatible phenotype and the isf fertility reduction with T  the N. crassa wild-type Oak-Ridge strain, and (b) if either of the two loci is identical to tof. Mutagenesis of the tof alleles was used as the first approach to tackle this dilemma.  5.2 MATERIALS AND METHODS 5.2.1 Strains, media and culturing methods The N. crassa strains used in the study are listed in Table 5-1. Strains 912-4, 912-10 and 912-72 contain het-c and tof loci introgressed from N. tetrasperma. They were obtained with T  the use of the following strains (Jacobson, 1992): N. crassa trp-4 (FGSC 4059 and 4060), arg-1 (FGSC 324) and leu-3 (R156 from D.D. Perkins); N. tetrasperma (FGSC 1270 and 1271); N. tetraspermalN. crassa hybrid C4,T4 (FGSC 1778; Metzenberg & Ahlgren, 1969). Culturing and crossing, using Vogel's (Vogel, 1964) and Westergaard's (Westergaard & Mitchell, 1947) media respectively, were performed as previously described, with modification (Davis & Deserres, 1970; Perkins, 1986). Mating tests were performed using/7 a (FGSC 4347) andflA (FGSC 4317) tester strains (Perkins et al., 1989). In a heterokaryon compatibility test, conidial suspensions from two strains (with complementary auxotrophic markers) are spotted on Vogel's minimal agar medium. Strains forming vigorous conidiating cultures after 3 days at 30°C are considered compatible.  138  Origin  Strain  Genotvpe  912-4  het-c (ili-2 ); arg-1; tof (ili-l  is?) A  D . J . Jacobson  912-10  het-c (ili-2 ); leu-3; to? (Hi- f  is?) a  D . J . Jacobson  912-72  het-c (ili-2 ); tof (ili-l  isf) trp-4; leu-3 a  D . J . Jacobson  85A  N. tetrasperma A  F G S C 1270  85a  N. tetrasperma a  F G S C 1271  R4-71  his-5 tol trp-4; pan-2 a  This study  R4-72  ade-3B; trp-4 cot-1; pan-2 A  This study  -  fla  F G S C 4347  Y2  thr-2 het-c  RT1-36  arg-5; het-c  (ili-2 ) a  N . L . Glass  RT1-40  arg-5; het-c  (ili-2 ) a  N . L . Glass  T  T  T  T  T  T  T  c  flA  F G S C 4317 het-6  0R  PA  PA  PA  hyg a  N . L . Glass  R  c  c  Table 5-1. List of Neurospora strains used in this study. ( F G S C = Fungal Genetics Stock Center). A l l strains are N crassa or N crassa introgressed strains except N. tetrasperma 85A and 85a. A superscript C or T specifies the wild-type allele origin from N. crassa or from N. tetrasperma, respectively. A l l strains are of N. crassa Oak Ridge compatible background, with the exception of introgressed strains (912-4, 912-10 and 912-72), which contain a mostly N. crassa background with introgressed N. tetrasperma loci (Jacobson, 1992). Strains designated with tol contain the original tol' allele (allele N 8 3 ; Newmeyer, 1970) while strains that do not have specification for tol allelism contain the wild-type to? allele (tof) from N. crassa.  N.  crassa strains that do not have designation for ili-1, ili-2, isi and het-c loci contain alleles  ili-l , c  ili-2 , isf and het-c , respectively. N. crassa het-c alleles (het-c ) used in this study include c  het-c  0R  0R  c  and het-c . In heterokaryon test, het-c PA  c is compatible with het-c  0R  T  PA  and het-c  PA  strain show incompatibility, but het-  but incompatible with het-c , and therefore displays het-c 0R  PA  specificity. 139  5.2.2 Repeat-induced point (RIP) mutation In N. crassa, duplicated sequences undergo methylation and GC—>AT mutation during mitotic divisions that precede karyogamy (Selker, 1990). In order to determine i f the tof locus is responsible for the sexual and non-allelic incompatibility phenotype, attempts were made to introduce R I P mutation at the tof locus. A n internal wild-type tol fragment from N. crassa (a 2.7 kb EcoRl fragment from D N A position 3-2735; Figure 3-3) was subcloned into a hygromycin-resistant vector (pCB1004; Carroll et al., 1994) and transformed into strain 912-4 het-c (ili-2 ) arg-1 tof (ili-l T  T  T  isf) A. Since N. crassa transformants are heterokaryotic and  contain both transformed and untransformed nuclei, a homokaryotic strain with only transformed nuclei was isolated from initial transformants according to Ebbole & Sachs (1990). The hygromycin-resistant homokaryotic transformants were then crossed to strain 912-71 het-c (iliT  2 ) tof(ili-l T  c  isf) trp-4 leu-3 a and T r p progeny were isolated. Progeny that gain sexual +  compatibility with either fl A or fl a tester strain (both wild-type N. crassa strains of Oak-ridge background) were subjected to Southern blot and R F L P analysis.  5.2.3 Nucleic acid isolation and hybridization Standard molecular biology procedures were used throughout (Sambrook et al., 1989). Genomic D N A isolation from Neurospora was adapted from Oakley et al. (1987). In the Southern blot analysis, three pg of D N A were digested with restriction enzymes and loaded in each lane; gel electrophoresis and nucleic acids transfer to N y l o n filters (Schleicher and Schuell, Keene, N H ) were performed according to manufacturer's specifications. [ a - P ] d C T P 32  (Amersham, Oakville, O N ) labeled probes were generated from digested D N A using the T7 QuickPrime K i t (Pharmacia, Baie d'Urfe, P Q ) .  140  5.2.4 tof isolation, plasmid construction and transformation Initially, fo/ -specific primers were used to isolate 5' and 3' fragments of tof in a P C R c  reaction from F G S C 1270 D N A . The primers used were tol A T G 1 (5' C C T G G G C T C A C C T A T G C 3'; base -50 to -34) and tol 11 (5' C C A G C A G T G G C T C A G C 3'; bases 1144 to 1129) for 5' fragment amplification and primers tol 17 (5' T A T A T G G G C G G C G A A G A T C 3'; Bases 1998 to 2016) and tol 21 (5' T G G A T G A T A T C A A G A C C T C A G C 3'; Bases 3317 to 3286) for the 3' fragment. The P C R amplification was performed in a model 480 Perkin-Elmer D N A cycler; P C R products were cloned into the P C R I I vector using the T A cloning kit (Invitrogen, San Diego, C A ) and subjected to D N A sequencing. The tof sequence revealed from the cloned 5' and 3' fragments were used to design ?cZ -specific primers to amplify internal regions. Many r  attempts failed to amplify the tof sequences corresponding to regions between bases 1144 and 1998 could not be amplified. However, in a P C R reaction involving primer tol 27 (5' G T C C T G C G C G A C T A A T C G C C G T 3'; Bases 1032 to 1053), mis-priming on the antisense strand occurred resulting in a tol 27-tol 27 amplification. The cloning and sequencing of the tol 27-tol 27 fragment revealed the missing portion of entire tof open reading frame.  Spheroplast preparation and transformation were performed as previously described (Schweizer et al., 1981; Vollmer & Yanofsky, 1986) with the exception of selecting for resistance to hygromycin B (250 units/ml; Calbiochem). Constructs of tof (see below) were assayed for T o l activity by co-transforming the cosmids (conferring hygromycin resistance) +  with a mat A-l-containing  p O K E 1 0 3 construct (pOKE103 has a pan-2 selectable marker; J.  Grotelueschen and R . L . Metzenberg, unpublished results) into strain R4-71 (his-5 tol trp-4; pan-  2 a) and strain R4-72 (ade-3B; trp-4 cot-1; pan-2 A) spheroplasts.  To obtain a clone containing the entire tof open reading frame, primers tol A T G 1 (5' C C T G G G C T C A C C T A T G C 3"; base -50 to -34) and tol 35T (5' C C C A A C G C T C C C T A T T G A T  141  G G A 3'; Bases 1770 to 1749 according to tof sequence) were used for P C R amplification. PCR-amplified product was cloned into the P C R I I vector using the T A cloning kit (Invitrogen, San Diego, C A ) and subsequently subcloned into pCB1004 vector which contains the hygromycin-resistant gene (Carroll et al, 1994) to create fo/ /pCB1004. Vector 7  to/ /pCB1004 7  was used to transform strain R4-71 in order to obtain transformants containing an introduced tof copy. Homokaryotic strains with only transformed nuclei were isolated from the initial transformant according to Ebbole & Sachs (1990). The to/7pCB1004 to R4-71 transformants were tested for their ability to trigger sexual incompatibility with a tof isf strain (fl A); fo/ /pCB1004 c  to R4-71 (containing 3.5 kb Spel-Nsil tot fragment; position -63 to 3407) and  pCB1004 to R4-71 transformants were used as controls.  5.2.5 DNA sequence analyses D N A sequences were determined using the A B I automated Taq DyeDeoxy Terminator cycle method (Mississauga, O N ) at the N A P S unit, Biotechnology Laboratory, University of British Columbia and at the D N A Sequencing Facility, University of California, Berkeley. Computer sequence analyses for protein and D N A were done using the M a c Vector/ A s s e m b l y L I G N software (International Biotechnologies, N e w Haven, C T ) and G C G package available from the Wisconsin Genetics Computer Group (Devereux et al, 1984).  5.3 RESULTS 5.3.1 RIP (repeat-induced point) mutation of tof T w o loci closely linked to tof introduced an unexpected phenotype when introgressed from N. tetrasperma to N. crassa (Jacobson, 1992). The ili-l  T  locus mediates non-allelic  incompatibility with ili-2 while the isf locus has allelic sexual incompatibility with isf. c  determine i f tof is equivalent to ili-l  T  To  and/or isf, I attempted to mutate the tof allele by R I P and  ask i f the two phenomena are still observed in tof mutants. 142  A tof internal D N A construct was transformed into strain 912-4 het-c (ili-2 ) arg-1 tof T  (ili-l  T  T  isf) A. Since N. crassa transformants are usually heterokaryotic and contain both  transformed and untransformed nuclei, homokaryotic strains with only transformed nuclei were isolated from initial transformants (Ebbole & Sachs, 1990). T w o homokaryotic transformants (912-431 and 912-451) were then crossed with strain 912-72 het-c (ili-2 ) tof(ili-l T  T  isf) trp-4  c  leu-3 a separately (912-451 Female x 912-72 Male; 912-431 M a l e x 912-72 Female). T r p  +  progeny were selected so that the potentially RIP-mutated, resident tof locus was preferentially recovered (trp-4* is closely linked to tof while trp-4' is linked to tof). Both crosses had a drastically lower number of mature perithecia, which is characteristic of a cross involving tof  (isf) and tof (isf).  Fifty T r p progeny were isolated from the two crosses ( V I - 1 to V I - 2 5 from 912-451 x +  912-72 and V2-1 to V2-25 from 912-72 x 912-431). Out of the 50 trp-4 progeny selected, 20 of +  them gained full fertility with either A tof (isf) or a tof (isf) partner (fertility of these 50 progeny, hereafter, is referring to their fertility with a tof isf partner). Table 5-2 summarizes the genotypes of the 50 progeny. Since trp-4* is closely linked with the tof locus in the 912431/912-451 parents, these progeny therefore represent potential tof-RYP mutants.  To test i f these potential tof-RIP mutants also lose their ability to mediate non-allelic incompatibility with ili-2 , some of the fully fertile progeny were tested in a heterokaryon c  compatibility test or in a progeny inhibition test (when forcing markers were not available) (D.J. Jacobson, unpublished results). In a progeny inhibition test, strain V l - 6 het-c (ili-2 ) arg-1 tof T  m p ?  (ili-l  T  T  isf) A, which is fertile with the tof (isf)fl a tester strain, was crossed with Y 2 (which  contains ili-2 ). c  The V l - 6 (tof' ) x Y 2 (ili-2 ) cross did not produce any inhibited progeny (all RIP?  c  48 germinated spores have w i l d type growth). This is in contrast with the control cross 912-4 (ili-l ) x Y 2 (ili-2 ), in which 1/4 of the progeny (10 out of 43 germinated spore) exhibit T  c  143  Strains  Markers  Vl-1  mat  Strains  a  V2-1  a poorly A poorly  Markers  mat  Vl-2  hyg  ?  V2-2  Vl-3  hyg  ?  V2-3  Vl-4  arg-1  A poorly  V2-4  Vl-5  hyg  a  V2-5  Vl-6  arg-1  A  V2-6  arg-1  a poorly  V2-7  arg-1 hyg  A  V2-8  arg-1 hyg  a  V2-9  R  R  R  Vl-7  a a poorly  leu-3 hyg  R  a A poorly A poorly  R  Vl-8  arg-1  Vl-9  hyg  Vl-10  arg-1 hyg  A poorly  V2-10  leu-3  a  Vl-11  arg-1  ?  V2-11  arg-1  A poorly  Vl-12  arg-1  A  V2-12  arg-1  Vl-13  hyg  ?  V2-13  leu-3 hyg  ?  Vl-14  arg-1  a poorly  V2-14  arg-1  A poorly  Vl-15  hyg  Vl-16  R  R  R  A poorly  R  ?  A poorly R  ?  V2-15  a  hyg  R  A  V2-16  a  Vl-17  hyg  R  a  V2-17  leu-3  Vl-18  hyg  a  V2-18  leu-3 hyg  a  V2-19  a  V2-20  arg-1  A poorly  a  V2-21  arg-1  A poorly  a poorly  V2-22  leu-3  ?  a  V2-23  leu-3 hyg  a  V2-24  leu-3  A  V2-25  arg-1 hyg  R  R  Vl-19 VI-20 Vl-21  hyg  R  VI-22 VI-23  arg-1 hyg  R  VI-24 VI-25  arg-1 hyg  R  ? ?  R  A poorly  ?  R  a poorly A poorly  R  Table 5-2. Genotypes of 50 progeny from the tof-RIP  cross. Progeny from the RIP cross were  tested for their auxotrophic markers, hygromycin-resistance and mating identity. A tof internal D N A construction was introduced into strain 912-4 het-c (ili-2 ) arg-1 tof (ili-l T  T  T  isf) A to  obtain transformants 912-451 and 912-431. The two hygromycin-resistant (hyg ) transformants R  were then crossed to 912-72 het-c (ili-2 ) tof(ili-l T  T  c  isf) trp-4 leu-3 a separately to obtain V I  and V2 progeny, respectively. All the progeny were selected on a Vogel's (1964) medium lacking tryptophan (hence selecting for typ-4 progeny, which is linked to tof). +  Twenty trp-4"  progeny confer full fertility when mated with tof (isf) testers. The rest show poor fertility with the tester strains; only a few (and sometimes none; as denoted by "?") mature perithecia can be observed in a 1-cm diameter mating-spot.  144  inhibited growth. It was concluded in the V I - 6 strain, activities for non-allelic incompatibility and for sexual incompatibility were absent. It is possible that R I P mutation of tof affects both ili-l  and isf function.  T  T w o fully fertile progeny, V 2 - 1 0 and V I - 2 3 (presumably tof ), R,p  were subjected to  incompatibility tests ( D J . Jacobson, unpublished results). Results show that V 2 - 1 0 formed a compatible heterokaryon with two ili-2 tester strains (RT1-36 and RT1-40). In contrast, the c  sterile control strain V2-17 (presumably not RIP-mutated) did not form stable heterokaryon with the two ili-2 tester strains. Therefore, like V I - 6 , both non-allelic incompatibility and sexual c  incompatibility are absent in the V2-10 progeny. However, in a heterokaryon compatibility test, fertile V l - 2 3 progeny behaved like V2-17, which did not form a vigorous heterokaryon with the two ili-2 tester strains. This is the first time that the non-allelic incompatibility activity (ili-l c  T  ili-2 interaction) is segregated from the sexual incompatibility activity (isf-isf c  the locus spanning ili-l  and isf. The interpretation is that in V l - 2 3 , isf activity is disrupted  T  while the ili-l  T  interaction) in  activity is intact.  So far, the experimental data suggested that in some of the tof  mp  10), both the ili-l  T  progeny ( V I - 6 and V 2 -  and isf activities were disrupted, whereas in another tof  RIP  progeny ( V l - 2 3 ) ,  only isf activity was affected. A tempting hypothesis to explain the above observation is that tof is equivalent to both ili-l  T  and isf locus and that it contains both non-allelic and sexual  incompatibility activity. In the case of V l - 2 3 , only the functional domain responsible for isf activity is disrupted, therefore making it fertile with an isf strain but incompatible with an ili-2  c  strain. However, it is possible that tof represents the isf locus only, and that the extension of the RIP-mutation process beyond duplicated sequences (Selker, 1991) may introduce mutations to the closely linked ili-l  T  locus in strains V l - 6 and V2-10.  145  However, complicating results occurred when V l - 6 and V2-10 (both lost sexual and nonallelic incompatibility; presumably tol ) TRW  were tested for mating-type incompatibility. Results  (data not shown) demonstrated that V l - 6 and V2-10 can only form heterokaryons with the same but not with the opposite mating-type, i.e. the potential tof  RIP  strains behave like a wild-type tof  allele in a heterokaryon incompatibility test. A normal tof allele behaves like a tol, which, when present i n both partners, allows stable heterokaryon formation between strains with opposite mating-types.  To understand the nature of the tof locus in the RIP-cross progeny, the 50 progeny and their parents were subjected for Southern blotting and R F L P analysis using tof sequences as a probe. A s expected, R F L P analyses showed that all the poorly fertile progeny have the same restriction pattern as their tof parent (912-72) (Figure 5-1A). However, for the fully fertile progeny, instead of having a tof RIP pattern, as expected, all progeny have a restriction pattern resembling their tof (912-72) parent (Figure 5-1A).  Since the trp-4* marker is closely linked to the tof allele (trp-4 is about 1 map unit from tol in N. crassa), it is possible that in these fully fertile trp-4 progeny, either conversion (i.e. tof +  is converted to tof) or a high frequency of recombination between tof (ili-l  T  isf) and trp-4 had +  occurred. The presence of a to/ -like R F L P pattern in the fully fertile progeny suggested the c  presence of tof allele in those strains, which could explain why fully fertile strains such as V l - 6 and V2-10 behave like a tof strains when tested in a mating-type heterokaryon incompatibility test.  For the conversion theory, we can hypothesize that sometime before the fertilization, a tof to tof gene conversion event has occurred in some of the transformed nuclei, using the integrated tof internal fragment as the donor D N A . Since the converted nuclei have a tof  146  Figure 5-1. RFLP analysis of tol sequences in the progeny from the to/ -RIP cross. A . The fertile r  (F) progeny isolated from the tof-RIP cross contain R F L P pattern similar to the tof allele (C) rather than to the tof allele (T). Number 1 to 25 referred to strains V2-1 to V2-25, which are progeny from the cross 912-431 (R) x 912-72 (C). Strain 912-431 (R) was constructed by introduction of a tof copy into strain 912-4 (T), which is of tof background. Three pg of each D N A was digested with restriction enzyme Hpall and was used for Southern analysis. The blot was probed with a 5.1 kb Sall-EcoRl to//pCJ31004 plasmid (see Figure 4-1). B. Same as panel A, except the hybridization blot was probed with pMOcosX cosmid X7:C2, which is mapped between tol and trp-4 (Figure 3-1). The fertile progeny show a N. crassa pattern (C).  147  sequence instead of tof (assuming that tof locus is equivalent to isf), they w i l l have a much higher fertility with an isf strain and therefore are much more representative in the progeny.  For the recombination theory, it is hard to explain why the introduction of the tof fragment would trigger such a high recombination rate between trp-4* and tof {ili-l  T  isf) as  observed i n the R I P cross. One hypothesis is that the introduced tof fragment, through an unknown mechanism, creates a recombination hot spot between trp-4* and tof (ili-l  T  isf). A n  alternative explanation is that somehow the trp-4* tof progeny have a selective advantage. For example, trp-4* tof progeny may have a faster germination rate than trp-4* tof progeny and are preferentially picked during ascospore germination.  To test i f crossovers between trp-4 and tol actually has occurred in the fertile progeny, D N A from the progeny were probed with cosmid X 7 : C 2 , which maps between trp-4 and tol ( X 7 : C 2 contains an overlapping region with X 2 5 : D 7 , a cosmid that contains tol; Figure 3-1). L i k e the previous hybridization result, the fertile progeny show a R F L P pattern resembling their tof (912-72) parent when probed with X 7 : C 2 (Figure 5-1B). This result agreed with the recombination theory showing that crossovers have occurred between trp-4 and X 7 : C 2 in the fertile progeny.  The analyses of the progeny did not give us further details on the isi locus, except to confirm that tof is closely linked to isf phenotype. W e cannot conclude or exclude the notion that tof is equivalent to isf.  However, the V l - 2 3 phenotype (which has a tof pattern and is  incompatible with ili-2 ) suggests it is unlikely that the tof locus is responsible for the c  ili-l  T  activity.  148  5.3.2 Molecular cloning and sequencing of tof Since the tof-RIP experiment does not provide unambiguous answers concerning the nature of the relationship between tof, ili-l  c  and isf, the molecular nature of tof needed to be  determined. If we know the sequence of tof, it would be feasible for us to determine i f the fully fertile progeny contained a converted sequence or not. Moreover, the cloning of tof would allow us to directly test (in a transformation and complementation assay) i f tof mediates sexual and non-allelic incompatibility.  Primers specific for tof were used to amplify fragments of the tof allele from the N. tetrasperma strain; primers specific for tof were later designed from the partial sequences and were used to amplify the tof O R F (see Materials and Methods). The tof allele contains a deletion of G at base 1244 (position according to tof sequence) as compared to tof; the singlebase deletion leads to a -1 frame-shift at amino acid position 384, changing the amino acid 384A to 384P-385S-386R-387 premature stop (amino acids position according to tof; the entire T O L  T  contains 384 amino acids, whereas T O L contains 1011 amino acids; see Figure 3-3). The c  truncated T O L contains the coiled-coil domain (position 177-211) but lacks the majority of the T  S E T domain (position 315-583) and the entire leucine-rich repeat (position 804-823). A l l three domains are required for T O L function, i.e. the ability to mediate vegetative incompatibility c  between opposite mating-types (see Figure 4-1). The amino-acid comparison between T O L  T  and  the amino terminal portion of T O L is shown in Figure 5-2. c  Since the molecular difference between tof and tof is revealed, it is possible for us to determine the molecular characteristics of the fully fertile progeny from the R I P cross. Characterization of progeny such as V2-10 (loss of both sexual and non-allelic incompatibility) and V I - 2 3 (loss of sexual but retention of non-allelic incompatibility) would allow us to determine i f a tof —> tof sequence is converted as predicted by the conversion model (in the  149  TOL-C Tol-T  TOL-C Tol-T  TOL-C Tol-T  10 20 30 40 50 60 MISPSSSCPTSSINSNTYPVSSSSASSPIPSSSHLDPESRSHKGSYEAVLEYFTSNSLPL MISPSSGCPTSSINSNTYTVSSSSASSPIPSSSHLDPDSRSHKGSYEAVLEYFTSNSLPL 10 20 30 40 50 60 70 80 90 100 110 120 PSYLQTYDSFVSHFFTSTDNPSLLSPINIPPHLTSYEAFIRYSIAKAAPAASSLPSTVPL PSYLQTYDSFVSRFFTSTDNPSSLSPINIPPHLTSYEAFIQYSITKAAPAAS-LPPTVPL 70 80 90 100 110 130 140 150 160 170 180 PLNQFCPRCTKALASSPPPPSHAAWWRRGGELCTPEELFRSASEGQCQFCWFVMVKVLRR PLNQFCPRCTKALAS SAPPRSHAAWWRRGGELCTPEELFHSASEGQCQFCWFVMVKVLRR 120 130 140 150 160 170 190 200 210 220 230 240 LWGVQIEDEEDLKQKVEEVWRDVKYLLVGLSFWAEDRREDVWRGEVSVRCVPRVPLGRE  TOL-C Tol-T 180  TOL-C Tol-T  LWGVQVEDEEDLKQKVEKVWRDIKYLGVALFFWAEDRREDVWRGEVSVRCVPRVPLGRE 190 200 210 220 230  250 260 270 280 290 300 KDVDVDMEGKVWSEFVRLRRGYIPGPSYPPSFLASFHTASEQSWSQALEWINNCRSHAL KDVDVDMEGKVWSEFVRLR-GYIPGPSYPSSFLASFHTASEQSWNQALEWINNCRSHAL 240 250 260 270 280 290  310 320 330 340 350 360 TOL-C CSAAETMDCHERRPARLIAVGRPGETHVRVIETAGLAVSETPFMSLSHCWGKDGVPTQLL Tol-T  TOL-C  CSAAETMDCHDRRPARLIAVGRPGETHVRVIETAGLAVSETPFMSLSHCWGKDGVPTQLL 300 310 320 330 340 350 370 380 KGNYDRFTKEGIRLTELPKTFRDAIE  T o l - T KENYDRFTKEGIKLTELPKTFRDPSR 360 370 380  Figure 5-2. A m i n o acid alignment between T O L The 384-aa T O L compared to tof  T  T  (N. tetrasperma) and T O L  c  (N. crassa).  was compared with the first 366-aa of T O L ; tof has a single base deletion as c  (at tof  D N A position 1244 G ) leading to a frameshift mutation (after T O L  T  aa  position 382) and a premature stop codon. The two polypeptides are 9 3 % identical over the TOL  T  O R F . ":" indicates a conserved amino acid, whereas "." indicates a conservative change.  The coiled-coil domain (position 177-211 according to T O L ) was underlined. c  150  case of V I - 2 3 , a partially converted sequence) or i f they contain complete tof sequence, which is a result of recombination.  The tol alleles from V2-10 and V l - 2 3 were amplified and subjected to D N A sequence analysis. The results showed that the tol alleles from these two strains contain the entire tof sequences. The tof sequences from these two alleles cannot be a product of tof —> tof conversion, since both of them contain the tof promoter region (instead of a tof promoter region, which contains a 3-bp deletion before the A T G start codon), and that the introduced tof copy is composed of internal tof region and does not contain any tof promoter sequences. These results agreed with data from the hybridization experiments (Figure 5-1) and support the model that the fully fertile progeny as generated in the R I P cross (see section 5.3.1) are products of recombination between tof and trp-4 and not due to a tof —»tof conversion event. Since V l 23 contained tof (and not tof) sequence and yet it retains non-allelic incompatibility with ili-2 , c  the hypothesis that tof is responsible for the ili-f  phenotype is refuted.  5.3.3 Transformation assay for tof non-allelic and mating-type incompatibility activity The tof allele (containing the entire tof O R F ) was PCR-amplified, cloned and subcloned into a Neurospora vector conferring hygromycin-resistance (pCB1004) to obtain  to/ /pCB1004. r  When the to/ /pCB1004 vector was transformed into recipient containing ili-2 background r  c  (strain R4-71), transformation frequency is similar to control pCB1004 vector (both have >500 transformants per plate), i.e. transformation reduction is not observed. The transformants from the tof/pCB1004 transformation do not show an inhibited phenotype (such as aconidiating and slow growing) as observed in partial diploids or transformants containing both antagonistic het genes (such as ili-l /ili-2 T  c  progeny). The transformation experiment therefore does not support  the premise that tof is responsible for the ili-l  T  phenotypes (i.e. incompatible with ili-2 locus) c  and that the two components (tof and ili-l ) may represent two linked but separate loci. This T  151  result is i n agreement with the phenotype of V l - 2 3 , which does not contain the tof allele (it contains tof instead) but still retains non-allelic incompatibility.  The tof allele was also tested for its ability to trigger mating-type-associated incompatibility. The to/ /pCB1004 vector was co-transformed with a construct containing mat 7  A-l into tol a spheroplasts. Transformation results showed that when co-transformed with mat A-l,  fo/ /pCB1004 7  does not trigger transformation reduction (88+15 transformants per plate); its  transformation frequency is comparable with an experiment using pCB1004 and mat A-l as transforming D N A (92±4 transformants per plate). The positive control (using ro/ /pCB1004 c  and mat A-l as D N A ) , on the other hand, induced at least a 20-fold reduction in transformation frequency when introduced into recipients containing tol a background (1+1 transformant per plate). These results suggested that the tof allele does not mediate incompatibility between mat A-l and mat a-l. This is in agreement with a previous experiment by Jacobson (1992), i n which he demonstrated that when tof is introgressed into the N. crassa background, opposite matingtype no longer mediates heterokaryon incompatibility. More over, it has been shown (see chapter 4) that all three functional domains (coiled-coil domain, S E T domain and leucine-rich repeat) are required for T o l function; truncation of T O L similar to the length of T O L also does +  c  T  not mediate mating-type incompatibility. The fact that T O L contains only the coiled-coil T  domain and not the entire S E T domain and the leucine-rich repeat (which is implicated in the incompatibility function in M A T A - l , M A T a - l and T O L ) may explain the lack of T o T function in N. tetrasperma. A presence of T o T function in N. tetrasperma w i l l unavoidably affect its pseudohomothallic lifestyle (N. tetrasperma is a self-fertile, natural heterokaryotic species). A s shown by Jacobson (1992), introgression of tof into N. tetrasperma leads to a mixed culture of segregated A and a mycelia instead of an A + a heterokaryon.  152  5.3.4 Testing of sexual incompatibility function in tof Although the phenotype of V l - 2 3 and the transformation assay does not support the premise that tof and ili-f between tof and isf.  are an identical locus, there is still no further data on the relationship  So far the two loci have not been segregated by genetic recombination. A s  a matter of fact, all the fully fertile progeny from the R I P cross also contain tof R F L P s , suggesting that the two loci are closely linked.  To determine i f the tof allele is responsible for sexual incompatibility between isf and isf, tof was introduced into a tol' background and the sexual phenotype of the transformants was observed. If tof is responsible for the isf activity, the transformants should gain sexual incompatibility with an isf strain. Vector fo/ /pCB1004 was used to transform a N. crassa tot r  strain (R4-71) in order to obtain transformants containing an introduced tof copy. The fo/ /pCB1004 r  transformants were then tested for their ability to trigger sexual incompatibility  with a tof isf strain. Transformants contain fo/ /pCB1004 D N A and transformants with vector c  only (pCB1004) D N A were also used as controls.  Twenty to/ 7pCB1004 homokaryotic transformants (from 11 individual transformants) 7  were isolated. For controls, 10 to/ /pCB1004 and 16 pCB1004 homokaryotic transformants c  (from 4 and 8 individual transformants, respectively) were isolated. Fifty p i of conidial suspension (roughly 10 /ml) from each transformant was spotted on thei7 A (tof 7  isf  background) tester strain (5 spots per plates). The ilf tol' recipient strain (R4-71) was used as a fully-fertile control while an isf strain (912-10) was used as a poorly-fertile control. Results from the mating assay showed that all the transformants showed normal mating reaction with full fertility comparable to the fully-fertile control (R4-71). O n the other hand, the poorly-fertile control (912-10) shows a typical poorly-fertile phenotype (roughly 20-fold less formation of mature perithecia) as expected from a reaction involving isf and isf as mating partners (see figure 5-3B for pictures of fertile and poorly-fertile crosses). 153  A Number of mature perithecia per 1 cm-diameter field  Strain L2-3 (tof  transformant)  47 ± 28.5  L I 1-2 {tof  transformant)  41.8 ± 4 . 4  L I 5-1 (tof  transformant)  58.6 ± 1 0 . 2  M 3 - 2 (tof transformant)  73.6 ± 2 0 . 1  M 5 - 2 (tof transformant)  83.4 ± 12.3  M17-1 (tof transformant)  65.8 ± 2 7 . 2  M20-1 (tof transformant)  64.6 ± 8.5  N2-2 (vector transformant)  49.2 ± 24.5  N5-1 (vector transformant)  70 ± 50.8  R4-71 (isf control)  45 ± 1 5 . 1  912-10 (isf control)  2.4 ± 1 . 7  B  Figure 5-3. Mating assay for tof transformants. ( A ) D N A from constructs tofZpCBl004, to/ /pCB1004, c  and p C B 1 0 0 4 (vector) were used to transform spheroplasts (strain R4-71) with  background tol isf.  Equal amount of conidia (1.25 x 10 ) from each transformant were used to 7  fertilize the tester strain fl A (which has an Uf background) on individual 8.5 c m petri dishes. R4-71 (isf) was used as a fully-fertile control while 912-10 (isf) was used as a poorly-fertile control. A typical mating reaction between i s l a n d isf contain roughly 20-fold less mature perithecia. For the counting of mature perithecia, five 1-cm diameter fields was randomly chosen from each plate (for the 912-10 strain, 10 fields were chosen) and the number of perithecia within the fields were counted. The right column represents the mean number of mature perithecia (with standard deviation) as seen in a field. (B) Pictures demonstrating mature perithecial production in a typical isf x isf (left panel) and a typical isf x isf (right panel) mating reaction (fields of 1 c m x 1 cm are depicted).  154  For a more quantitative assessment of the sexual phenotype of the transformants, equal amounts of conidia from each selective homokaryotic transformants were used in a mating test, and the number of mature perithecia was recorded. The results (Figure 5-3) showed that when mated with a isf partner, all the experimental transformants and the fully-fertile control (R4-71) had a roughly 20-fold margin of fertility over the isf control (912-10). These data showed that the transformation of the tof allele into an isf strain does not effectively convert it to a strain conferring an isf phenotype. Therefore, it is unlikely that the tof gene is responsible for the isf sexual incompatibility activity and that the two most probably represent two separate loci.  5.4 DISCUSSION The mating-type genes in N. crassa provide seemingly opposite functions during sexual and vegetative phase. The M A T A - l and M A T a-1 are important for conferring mating identity during cell-cell recognition in the sexual fusion event. Yet, during vegetative phase, the two proteins restrict heterokaryon formation between individuals of alternative mating-types. One key player in the restriction of heterokaryosis is the tol locus - a wild-type tol gene product is required for recognition, and eventually, rejection of opposite mating-type nuclei in a common cytoplasm.  N. tetrasperma, a pseudohomothallic species, is effectively self-mating; however, unlike homothallic species, it contains two distinct types of nuclei, A and a. After fertilization, four spores are produced in each ascus, in which both mating-types are packaged in the same spore. N. tetrasperma is therefore a natural A + a heterokaryon upon germination. The co-existence of the opposite mating-types during the vegetative phase is due to the inactive tof allele. In a series of introgression experiments, Jacobson (1992) showed that tof cm suppress mating-type incompatibility i n N. crassa whereas introduction of tof into N. tetrasperma leads to a breakdown of the A + a heterokaryon.  155  During the introgression of tof into N. crassa, Jacobson (1992) encountered two unexpected observations. Firstly, the tof trp-4* introgression progeny consistently show reduced fertility (less mature perithecia and slower development) as compared to tof trp-4 siblings when crossed to various tof trp-4* strains. This fertility reduction can by suppressed by a mutation in mat a-l (a ), which suppresses mating-type incompatibility but does not affect mating function m33  (Griffiths & DeLange, 1978; Staben & Yanofsky, 1990) (D.J. Jacobson, unpublished data). Since the sexual incompatibility factor (tentatively named isi) maps closely to tol and its effect can be eliminated by a suppressor of mating-type incompatibility, there is a possibility that the tol locus may in fact mediate the phenomenon. It is possible that the tof-tof  interaction  somehow prevents the switch off of mating-type incompatibility, which is normally turned off during sexual development. Alternatively, tol may play an active role i n the sexual phase and that the tof-tof interaction may interfere with its sexual function.  The second complication observed during tof introgression is the presence of a class of inhibited progeny showing phenotypes resembling strains with a partial duplication heterozygous for a het gene. The presence of inhibited progeny is commonly found i n P. anserina when the two mating partners contain antagonistic alleles of non-allelic het loci (see section 1.4). Further mapping for the antagonistic activity showed that the incompatibility is linked to tof and het-c  c  (see section 1.4 for het-c locus); the two loci were named ili-l  T  and ili-2 (D.J. Jacobson, c  unpublished data). Since both tol and het-c are involved in mediating heterokaryon incompatibility, the newly found non-allelic interaction may indicate that an unusual interaction between the two may lead to the triggering of incompatibility. These observations have an implication that tol and het-c may be involved in the same pathway.  Mutagenesis of tof using the R I P mechanism was used as a first approach to determine whether tof represents ili-l and isf activity. A copy of tof allele was introduced into a tof T  156  strain, in an effort to introduce RIP mutations to that allele. Although some progeny lost both jsi -mediated sexual incompatibility and r  //-mediated incompatibility, genetic characterization,  RLFP analysis and sequencing of some of the tol alleles suggested that these progeny do not representto/ -RIPmutants, but rather are genetic recombinants between the selected trp-4* r  marker and the tof ili-l  T  isf loci. The failure to recover tof-RIP mutants may be due to the fact  that the tof allele is not homologous enough to trigger RIP mutagenesis. Alternatively, since the frequency of RIP is largely dependent on the gene and the location of the resident copy (Irelan et al., 1994), it is possible thatto/ -RIPmutants can be recovered if more progeny are screened; r  only a 10 % success rate was observed when the same tof fragment was used to RIP-mutate the tof gene (see Chapter 4).  The recovery of such a high frequency of recombinants (40 %) between trp-4* and tof I Hi-1 Iisf was unexpected. Previous genetic crossing experiments suggested a close linkage T  between trp-4 and tollili-llisi (see section 5.1). Since progeny were germinated on a medium lacking tryptophan, it is possible that progeny with the trp-4*-\mkcd region from the tof parent coupled with theto/ -linkedregions from the tof parent have an unknown advantage and c  germinated better. Alternatively, the procedure of making spheroplasts coupled with the introduction of a tof fragment may have somehow increased recombination around the tof region through an unknown mechanism.  Althoughfo/ -RIPprogeny were not isolated, a recombinant (Vl-23) progeny containing r  tof isf ili-f background was recovered. This is the first time that ili-1 has been separated from the tol and isi locus. Since Vl-23 contains the tof allele, we can therefore eliminate the notion that tof is responsible for the ili-l  T  activity and conclude that tol and ili-1 represent two distinct  loci. Since Vl-23 contains a cross-over between ili-l  T  and tolltrp-4, this strain will be useful in  the future for mapping the precise location of the ili-1 gene using the cosmid walk that spans trp-  157  4  and tol (see Chapter 3) as probes. Preliminary data suggests that ili-1 maps between trp-4 and  cosmid X7:C2 (see Figure 3-1).  The tof allele was cloned in order to determine if it contains isf activity when transformed into a won-isf background. As shown by the mating assay, all to/ -transformants r  remained sexually compatible with a partner containing isf background. These results do not support the notion that tof is involved in the isf-isf sexual incompatibility reaction. Moreover, when an isf strain was crossed with various tof-RIP strains, reduction in fertility was still observed, demonstrating that tof is also unlikely to be involved in the sexual incompatibility reaction (D.J. Jacobson, unpublished results). Therefore, although tol and isi are closely linked, they are likely to be two separate loci. Nonetheless, we cannot discount the possibility of tof containing isf activity with absolute certainty. For example, it is possible that the introduced tof allele did not function properly as a transgene, hence explaining the absence of isf activity in the tof transformants. Mechanisms that interfere with expression of transgenes have been described in N. crassa (Cogoni & Macino, 1999).  Originally, it was thought that the sexual incompatibility between tof trp-4* strains and tof  trp-4  strains is due to the allelic interaction between tof and tof. However, the above  results have forced us to renew our model to better suit all the data collected. One possible scenario is that the isi gene product(s) may have an active role in the mating process of AT. crassa. The fact that a mutation in the mat a-l gene can suppress the sexual incompatibility between alternative isi alleles hints that the isi gene product may interact with the mating-type proteins.  Mating-type genes seem to be conserved among different Neurospora (and even related ascomycetes such as P. anserina) species and can function to confer mating activity in heterologous systems (see section 1.3; Shiu & Glass, 2000). However, despite conservation of 158  mating-type gene function, interspecies mating between closely related Neurospora species are either impossible or highly infertile. It is possible that the divergence of mating-modifiers such as the i5i locus may act to restrict interspecies mating and contribute to speciation. If the isi locus does have an active role in mating and participates in speciation, one prediction is that the isi locus should have a strict separation between Neurospora species that have more divergent mating strategies. Until we have fully characterized the nature and the function of the isi locus, we cannot assess whether it has a significant biological role in preventing interspecies mating.  This study provides evidence that ili-l , a locus involved in non-allelic incompatibility, is T  a distinct locus from tof. Non-allelic heterokaryon incompatibility has been implicated in fungi such as P. anserina and C. parasitica (Begueret et ah, 1994; Anagnostakis, 1982b). The antagonistic alleles for the het-clhet-e system in P. anserina have been characterized; they encode a glycolipid transfer protein and a GTP-binding protein, respectively (Saupe et ah, 1994; Saupe et ah, 1995a; see section 1.4). Since glycolipids have been implicated in the stress response during cell death (both apoptotic and non-apoptotic), it has been proposed that het-c and het-e may act as components of a signaling pathway that triggers incompatibility reaction (inhibition of growth, cell death, etc.) (Saupe, 2000). Interestingly, two other genes involved in non-allelic incompatibility in P. anserina, het-d (mediates het-c/d interaction) and het-v (mediates het-rlv interaction), are highly homologous to the het-e gene (J. Begueret, personal communication). Moreover, modifier mutations {mod-A mod-E) that suppress all three nonallelic incompatibility systems in P. anserina have been described (see section 1.4.8). These data suggest a close relationship between the different non-allelic incompatibility systems in P. anserina. Molecular characterization of ili-1 and ili-2 will show whether these two genes have sequence (and functional) homology to the non-allelic het genes in P. anserina.  Although allelic heterokaryon incompatibility systems have been well characterized in N.crassa (see section 1.4), non-allelic incompatibility has not been broadly studied; het-6159  associated incompatibility is the only het system in N. crassa that is speculated to be mediated by two distinct loci (Smith et ah, 2000b). In Neurospora, abortive and inviable progeny are commonly observed in crosses involving natural (non-laboratory) strains. It is possible that the low viability of progeny from these crosses is due to the effect of non-allelic incompatibility systems that have been overlooked. If non-allelic incompatibility does exist i n N. crassa, it w i l l affect the success rate of outbreeding in a species that is obligatory non-selfing. Alternatively, it is possible that the non-allelic incompatibility observed in the ili-llili-2  system is strictly  between different species (N. crassa and N. tetrasperma in this case) and not within a species. Extensive out-breeding and selective preference for viable progeny i n N. crassa may effectively eliminate polymorphisms at non-allelic incompatibility loci; many allelic incompatibility systems already exist in N. crassa to serve the function of nonself recognition.  Characterization  of ili-1 and ili-2 from natural N. crassa strains w i l l definitely shed light on this dilemma.  160  6. Concluding Remarks Although the two mating-type genes in N. crassa, mat A-l and mat a-l, confer mating identity for compatible sexual fusion, they mediate heterokaryon incompatibility during the vegetative phase. This aim of this study was to delineate the mechanism involved in mating-type mediated heterokaryon incompatibility in N. crassa. T w o separate but connected approaches were conducted to tackle the dilemma. Firstly, the mating-type gene mat A-l was functionally dissected for its heterokaryon incompatibility and mating ability. Secondly, cloning and characterization of tol was performed in order to understand its role i n mediating mating-type incompatibility.  Dissection of the mat A-l functional domain indicated that mating and incompatibility can be spatially separated (Chapter 2). Mating function of mat A-l (and mat a-l) seem to be related to their roles i n transcriptional regulation. This can be shown by the importance of a( M A T A - l ) and H M G (mat a-l) domains in mating function. However, data from this study on mat A-l (and previous studies on mat a-l) suggest that protein-protein interaction domains (i.e. leucine-rich repeat, L R R ) are important in the mediating mating-type incompatibility. In support of this theory, preliminary yeast 2-hybrid data from C . Staben (University of Kentucky) showed that the M A T A - l and M A T a - l mating-type proteins physically interact. The interaction between the two mating-type proteins appears to be important for heterokaryon incompatibility but not for mating. A mutation in mat a-l (a deletion in the L R R region), which abolishes mating-type incompatibility but does not affect mating function, eliminated physical interaction between M A T A - l and M A T a - l . It is possible that the M A T A - l leucine-rich repeat identified in this study may facilitate the physical interaction of the two mating-type proteins, which in turn represents the molecular basis of recognition between individuals containing antagonistic mat genes.  161  Although now that we have a model for recognition of alternative mat alleles, little is known about the pathway or biological actions that the recognition may trigger. One way to study the mechanism involved in the incompatibility reaction is to delineate its mediator(s). The tol locus was first identified as a suppressor mutation for the mating-type incompatibility. In this study, the tol gene was cloned and characterized (chapter 3 and 4). The tol gene is not a homolog to any known genes. It contains two protein-protein interaction domains (coiled coil and LRR) and a region of similarity to two het genes (het-6 and het-e). The discovery of a common domain shared by different genes involved in the incompatibility reaction is encouraging. It may hint that although different het systems encode functionally diverse proteins (chapter 1), they may share a common biochemical pathway (cell death pathway). Microscopic studies have shown that all the het systems tested so far show similar responses during an incompatibility reaction (Chapter 1). How tol mediates the incompatibility between alternate mating-type proteins is still a dilemma. However, data presented here hint that a direct interaction between the mating-type proteins and the tol gene product is possible (Chapter 3). In support for the model for a TOL/A-l/a-1 multimerization, a weak physical interaction has been detected between TOL and MAT A - l (but not between TOL and MAT a-1) by yeast two-hybrid system (C. Staben, T. Badgett and P. Shiu, unpublished data).  Since the tol gene product does not contain any identifiable domains as shared by other proteins with known biochemical function, we have little clues for the biochemical mechanism that mediate incompatibility. However, the presence of two protein-protein interaction domains suggested that TOL may act as an adapter protein for formation of a larger protein complex. Attempts have been made to isolate proteins that interact with TOL (Chapter 4). One of the top genes (TOL-interacting protein) identified encodes a protein that could be involved in cell cycle control (VIPl). It is possible that multimerization between VIPl and the TOL/MAT A-l/MAT a-1 complex may trigger a pathway similar to apoptosis (programmed cell death).  162  Additional genetic studies around the tol locus have identified an additional locus (isi) that is involved in mating, and possibly mating-type associated incompatibility (Chapter 5). What we know so far for the newly identified locus is that its alternative alleles mediate sexual incompatibility and that incompatibility can be suppressed by mutations that suppress matingtype-associated heterokaryon incompatibility. Sexual incompatibility as mediated by the isi locus may represent an extension of heterokaryon incompatibility to the sexual phase. Its association with tol is still unclear, and there is no evidence that the two gene products act in the same pathway. Although it is possible that previously identified suppressors for mating-type incompatibility, which map closely to tol, may in fact be due to mutation in the isi locus ; we cannot rule out the possibility of a gene cluster that controls both mating functions and matingtype incompatibility.  This study has provided clues about the mechanism involved in the mating-typeassociated incompatibility. The model we have is premature, but the data collected so far has opened the opportunity for future experiments to address the questions that we may have generated in the course of this study, such as the biological significances of the top genes, the biochemical function of the S E T domain, the localization of T O L and the mating-type proteins, the mechanism of tol repression during sexual cycle, the molecular characterization of tol alleles in various species and their relations to their sexual strategy and the mechanism of recognition and triggering of cell death. 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(1999) Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors. Proc. Natl. Acad. Sci. USA 96: 5592-5597. Zickler D., Arnaise S., Coppin E., Debuchy R. & Picard M. (1995) Altered mating-type identity in the fungus Podospora anserina leads selfish nuclei, uniparental fungus, and haploid meiosis. Genetics 140: 493-503.  206  8. Appendices 8.1 Genetic crossover between his-5 and tol can be detected by cosmid G13:C8  O  T4  Til  T18  T21M  Figure 8-1. Genetic crossover between his-5 and tol as detected by G 1 3 : C 8 . Progeny containing genetic crossover between his-5 and tol was probed with probed with D N A from cosmid G13:C8 (see Figure 3-2B). Since the parental strains contain different R F L P pattern on L G I V , it is possible to detect a crossover point i f a particular cosmid is located between his-5 and tol. When probed with cosmid G 1 3 : C 8 , progeny T4, T18 and T21 show a R F L P pattern of the Mauriceville parent ( M ) (strain R5-28), demonstrating that the recombination point must occurred between G13:C8 and the last cosmid walk (X25:D7; which detect only Oak Ridge pattern in all the progeny). Progeny T l 1 shows a hybrid pattern resembling both the Mauriceville ( M ) (R5-28) and the Oak Ridge (O) (R5-27) parents, demonstrating that the crossover point in this progeny must be within the cosmid G13:C8.  207  8.2 physical map of cosmids containing the tol gene  G13:C8  Mx»l 2179 bp  tol tol EcoRI (v)/  Spel •63 bp  Mbo\ -2kb  Afe/1 3400 bp  EcoRI 4.9 kb  Sah 0.8 kb  X25:D7  Figure 8-2. Physical map for cosmids X 2 5 : D 7 and G13:18. A 6.9-Kb EcoRI-EcoRI fragment from cosmid X 2 5 : D 7 was determined to contain T o l activity in a co-transformation assay (see +  section 3.2.4). A 4.2 kb Sall-Nsil fragment was later subjected to D N A sequencing. D N A analysis also shows that G13:C8 contain partial sequence for the tol ORF. EcoRI (v) denotes a restriction site present in the multi-cloning site i n the p M O c o s X cosmid (Orbach, 1994).  208  8.3 Hydrophobicity Plot for T O L  •? o  •g.  I.OOo . o o -—i  6. °  ^  i?  -1.00-  -2.00-  S c a l e = Kyte-Doolirtle  Hydrophilicity Window S i z e =  5.004.003.002.00-  1—t  • II.M. i i >ily •  -1—t—t  I 11  1 j 1  i  !  _ J _ — l _ J  L.  1  n  )fl li  -3.00-  f  i  t  '  IT™T*< 1  -1  —  •  -4.00-5.00-  200  400  600  800  1000  Figure 8-3. Hydropathy plot of T O L as analyzed by the method of Kyte and Doolittle (1982) with a 7-residue window. Hydrophilic regions correspond to the positive index numbers. T O L has a predicted isoelectric point of 4.67 and is made up o f 40% non-polar and 26% charged residues. The carboxyl-terminal portion of T O L (amino-acids position 837 to 1011) is relatively hydrophilic, composed of 30% non-polar and 30% charged residues.  209  8.4 Detection of vipl sequence in various Neurospora species  Figure 8-4. Detection of vipl sequences i n various Neurospora species. Genomic D N A was digested with E c o R I and probed with clone 7D8 (N. crassa vipl homolog). Species names and the presence of mating-type sequences are given (A+a = contains both mat nuclei; Pbggeler, 1999); A/a = homokaryon with both mat genes). A m o n g the Neurospora species listed here, N. crassa and N. intermedia are the only species that confer mating-type-associated incompatibility. N. sitophila and N. tetrasperma are mating-type compatible species with a tollike suppression (Perkins, 1977; Jacobson, 1992). The crassa strains used are #43 (tol) and 74O R 2 3 - I V A (wild type). Other species used are listed in Table 4-1. 210  

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