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Non-self recognition in filamentous fungi : the het-c mediated vegetative incompatibility in neurospora… Wu, Jennifer Donglan 2000

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N O N - S E L F R E C O G N I T I O N I N F I L A M E N T O U S F U N G I - T H E het-c M E D I A T E D V E G E T A T I V E I N C O M P A T I B I L I T Y I N NEUROSPORA CRASSA by J E N N I F E R D O N G L A N W U B . Sc., Peking University, 1988 M . Sc., Peking University, 1991 A THESIS S U B M I T T E D FN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Biotechnology Laboratory and Department of Botany We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A March, 2000 © Jennifer Donglan W u , 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date vAcrcUa\, Z-OOO DE-6 (2/88) Abstract In Neurospora crassa, at least eleven het loci including the mating-type locus have been identified that regulate non-self recognition during vegetative growth. One of the genetically and molecularly characterized loci , het-c, was shown to encode three alleles het-cOR, het-cPA and het-cGR. The three het-c alleles encode similar polypeptides with a 34 - 48 amino acid polymorphic region that controls allelic specificity. In an effort to understand the biological role o f the het-c locus in filamentous fungi, the sequences o f het-c specificity region from species within Neurospora and its related genera were analyzed. The het-c locus exhibited a trans-specific mode of evolution (an allele from one species is more closely related to an allele from another species or genus than to other alleles from the same species) based on a phylogenetic analysis of D N A sequences and an increased frequency and number of nonsynonymous nucleotide substitutions in the polymorphic domain. This study suggested that the het-c locus is under balancing selection for the function of mediating non-self recognition. The polymorphic region in the peptides encoded by the three het-c alleles was dissimilar by both amino acid sequences and the pattern o f deletion or insertion. To differentiate whether the composition of amino acid sequences or the pattern of insertion/deletion is the critical determinant for het-c allelic specificity, chimeric constructs of naturally occurring and artificially generated het-c alleles were introduced into N. crassa strains with alternative het-c specificities. Incompatibility of the transformants was assayed by occurrence of dead hyphal compartments, growth rate and colony morphology. This study suggested that spatial characteristics as affected by the pattern and size of the deletion/insertion within the specificity domain were the primary determinant for het-c allelic specificity. Immunoprecipitation studies indicated that non-self recognition is mediated by H E T - c heteromeric complex formation during vegetative incompatibility, and that differences in the specificity domain affect the capacity to make heterocomplexes versus homocomplexes. ii Table of Contents Abstract ii List of Figures List of Tables Acknowledgements Dedication xi Chpater 1 General Introduction 1 1.1 Self and non-self recognition 1 1.1.1 Self and non-self recognition in mammals 2 1.1.1.1 Sexual recognition 2 1.1.1.2 Somatic incompatibility - the MHC complex 2 1.1.2 Sexual incompatibility in plants - the S locus 5 1.1.3 Self and non-self recognition in ascomycete fungi 8 1.1.3.1 Mating-type and mating recognition in ascomycetes 9 1.1.3.2 Vegetative incompatibility in filamentous ascomycetes 10 1.2 Molecular aspects of mating recognition in ascomycetes 13 1.2.1 Mating recognition in Saccharomyces cerevisiae 13 1.2.2 Mating recognition in filamentous ascomycetes 16 1.2.2.1 Life cycle of filamentous ascomycetes 16 1.2.2.2 The mating-type locus 18 1.2.2.3 Pheromone response pathway 18 1.2.2.4 High-Mobili ty-Group ( H M G ) and a l domain proteins 19 1.3 Vegetative incompatibility in filamentous ascomycetes 20 1.3.1 Vegetative incompatibility in Podospora anserina 20 1.3.2 Vegetative incompatibility in N. crassa 24 1.3.2.1 Neurospora crassa, the model organism 25 1.3.2.2 Cellular response to vegetative incompatibility 26 1.3.2.3 Mating-type mediated vegetative incompatibility 28 1.4 het-c mediated vegetative incompatibility 30 1.5 Thesis objectives ...33 Chapter 2 Evolutionary analyses of the het-c locus 35 2.1 Introduction 35 2.2 Materials and Methods 44 2.2.1 Strains and media 44 2.2.2 Genomic D N A isolation and Southern analysis 44 2.2.3 P C R amplification of het-c specificity region 47 2.2.4 Subcloning of P C R products 47 2.2.5 Sequencing o f P C R products 49 2.2.6 Sequence analysis 49 2.2.7 Phylogenetic tree reconstruction 50 2.2.8 Calculation o f synonymous and nonsynonymous nucleotide substitutions 51 2.2.9 Statistical analysis 52 2.3 Results 53 2.3.1 Genomic Southern blot analysis 53 2.3.2 P C R products and D N A sequences 53 2.3.3 Amino acid sequences of het-c specificity region 55 2.3.4 Trans-specific polymorphism of het-c 60 2.3.5 Number o f synonymous and nonsynonymous substitution per site in het-c specificity region 73 2.4 Discussion 77 2.4.1 Trans-specific polymorphisms at het-c 77 2.4.2 het-c allelic lineages were generated prior to the divergence o f modern species 79 2.4.3 het-c allelic lineages 80 2.4.4 Possible mechanisms of balancing selection at the het-c locus 82 2.4.5 A n excess of nonsynonymous nucleotide substitutions in the het-c polymorphic region 83 2.4.6 het-c shares evolutionary features with MHC and S loci 84 Chapter 3 Molecular basis of het-c allelic specificity 86 3.1 Introduction 86 3.2 Materials and Methods 88 3.2.1 Strains and media 88 3.2.2 Recombinant D N A techniques 89 3.2.3 Isolation of naturally occurring het-c alleles 92 3.2.4 Generation of artificial het-c alleles 92 3.2.5 Generation of vector cassette for construction of chimeric alleles 98 iv 3.2.6 Constructs of chimeric alleles 100 3.3.7 Cloning chimeric constructs into N. crassa transformation vectors 103 3.2.8 Construction of strain CJ44 103 3.2.9 D N A transformation o f N. crassa 106 3.2.10 Heterokaryon incompatibility test 107 3.2.11 Growth rate measurement 107 3.2.12 Light microscopy 107 3.3 Results 109 3.3.1 Phenotypes of transformants with wi ld type het-c alleles 109 3.3.2 Amino acid sequence variability o f het-c alleles 113 3.3.3 Chimeric construction and specificity of het-c0R - type alleles 114 3.3.4 Chimeric construction and specificity of het-cGR - type alleles 117 3.3.5 Chimeric construction and specificity of het-cPA - type alleles 119 3.3.6 Summary o f the specificity of naturally occurring alleles 124 3.3.7 Artif icial PA-type alleles with size variations in the PA-specific insertion 125 3.3.8 Generation of artificial alleles with both PA-specific and OR-specific insertions 132 3.3.9 Alleles with chimeric combinations o f amino acid sequence and patterns o f insertion/deletion 137 3.3.10 Summary o f het-c specificity o f artificial alleles 139 3.3.11 Amino acid sequences affecting het-c activity 140 3.3.12 Co-transformation of alleles with new specificity 142 3.4 Discussion 146 3.4.1 Variation in het-c mediated incompatibility phenotypes 146 3.4.2 het-c allelic specificity was not primarily affected by amino acid substitutions in the variable region 148 3.4.3 The pattern and size of insertion or deletion in the variable region are the primary determinants for het-c allele specificity 150 3.4.4 Predicted protein structural characteristics of the hypervariable region 151 3.4.5 Mode l for generation of het-c allele specificity 154 3.4.6 The use of artificial het-c alleles to study protein-protein interactions 159 Chapter 4 Heterocomplex of alternative HET-c polypeptides 160 4.1 Introduction 160 4.2 Materials and Methods 163 4.2.1 Strains and media 163 4.2.2 Antibodies 163 4.2.3 Construction o f fusion genes 164 4.2.4 Preparation of cell extraction 171 4.2.5 Immunoprecipitation 171 4.2.6 SDS Polyacrylamide Gel Electrophoresis 172 4.2.7 Western blotting 173 4.3 Results 174 4.3.1 Fusion gene constructs 174 4.3.2 het-c activity o f fusion genes 176 4.3.5 Heteromeric complex formation of H E T - c proteins 177 4.4 Discussion 185 4.4.1 Non-self recongnition is mediated via protein heteromeric complex formation 185 4.4.2 Possible mechanisms for heteromeric complex formation 189 Chapter 5 Concluding Remarks 196 5.1 Summary 196 5.2 General model of how het-c mediates vegetative incompatibility 197 5.3 Future directions 200 6. Bibliography 202 7. Appendices 221 vi L is t of Figures Figure 1-1. Barrage zone between vegetative incompatible fungal strains 11 Figure 1-2. The mating process in Saccharomyces cerevisiae 15 Figure 1-3. The life cycle of N. crassa 17 Figure 1-4. Evan's blue staining of dead hyphal compartments 27 Figure 1-5. Ultrastructural characteristics of wi ld type and incompatible heterokaryons 28 Figure 1-6. Morphology of compatible and incompatible heterokaryons 30 Figure 1-7. Features of deduced H E T - c peptides from D N A sequences 32 Figure 1-8. Amino acid sequence comparison of the variable region II in H E T - c 32 Figure 2-1. Examples of trans-specific polymorphism 38 Figure 2-2. Schematic diagram illustrating the effect of balancing selection 43 Figure 2-3. Diagram of the primer locations for P C R amplification of het-c specificity domain 48 Figure 2-4. Southern hybridization o f genomic D N A from different species and genera with het-cOR probe from N. crassa 54 Figure 2-5. Inferred amino acid sequences of P C R amplified het-c allelic specificity motif from 40 isolates 58 Figure 2-6. Unrooted neighbor-joining tree from the D N A sequence alignment in the het-c specificity region including deletion/insertion sites 64 Figure 2-7. Unrooted neighbor-joining tree from the D N A sequence alignment in the het-c specificity region excluding deletion/insertion sites 65 Figure 2-8. Unrooted maximum parsimony tree from the D N A sequence alignment in the het-c specificity region including deletion/insertion sites 66 Figure 2-9. Unrooted maximum parsimony tree from the D N A sequence alignment in the het-c specificity region excluding deletion/insertion sites 67 Figure 2-10. Unrooted maximum likelihood tree from the D N A sequence alignment in the het-c specificity region including deletion/insertion sites 68 Figure 2-11. Unrooted maximum likelihood tree from the D N A sequence alignment in the het-c specificity region excluding deletion/insertion sites 69 Figure 2-12. Unrooted neighbor-joining tree of het-cPA - type alleles 70 Figure 2-13. Unrooted neighbor-joining tree of het-cOR - type alleles 71 Figure 2-14. Unrooted neighbor-joining tree o f het-cOR - type alleles 72 Figure 2-15. Frequency distribution o f nonsynonymous and synonymous nucleotide substitution along the codon position of het-c specificity domain 75 Figure 3-1. Amino acid sequences of the specificity region o f naturally occurring het-c alleles 90 Figure 3-2. P C R generation of recombinant het-c alleles 94 Figure 3-3. P C R generation o f het-c alleles by deletion 95 Figure 3-4. Conserved restriction sites in het-c used for chimeric construction 99 Figure 3-5. Principals for chimeric construction and transformation assay for het-c specificity 101 Figure 3-6. Plasmid map of the vector pCB1004 104 vii Figure 3-7. Plasmid map of the vector pOKE103 105 Figure 3-8. Colony morphology of transformants containing the wild-type het-c alleles into het-cOR, het-cPA and het-cGR background I l l Figure 3-9. Average growth rate of het-c transformants 112 Figure 3-10. Hyphae morphology and occurrence o f compartmentation o f the three classes of het-c transformants 112 Figure 3-11. Representative morphology o f transformants of naturally occurring mutant het-cOR chimeric constructs 116 Figure 3-12. Hyphal compartmentation and death ofNdi5923 (p26) chimeric construct Introduced into different het-c strains after 1 day and 3 days o f growth 124 Figure 3-13. Amino acid sequences comparison of H E T - c P A , P26 and P26m specificity region 125 Figure 3-14. Phenotype comparison of het-cPA,p26, p26m transformants after three days o f vegetative growth on Vogel 's selective medium 126 Figure 3-15. Average growth rate comparison of het-cPA,p26, p26m transformants 126 Figure 3-16. Amino acid sequence comparison of H E T - c A , H E T - c 0 R , H E T - c G R , P D 1 , PD2 , PD3 and PD4 specificity region 127 Figure 3-17. Average growth rate comparison of P A , G R , pdl, pd2, pd3 and pd4 transformants ofhet-cOR, het-cPA and het-cGR background 130 Figure 3-18. Phenotypes of vegetative growth of allele pdl, pd2, pd3 and pd4 transformants 130 Figure 3-19. Hyphal morphology and occurrence of dead hyphal compartments in transformants when pdl, pd2, pd3 and pd4 chimeric constructs were introduced into het-cOR, het-cPA and het-cGR spheroplasts 131 Figure 3-20. Amino acid sequence comparison of P O l and P 0 2 133 Figure 3-21. Phenotype of transformants of alleles pol and po2 chimeric constructs 135 Figure 3-22. Average growth rate o f alleles pol, po2 chimeric construct transformant 135 Figure 3-23. Hyphal compartmentation and death by the introduction o f pol, po2, chimeric constructs into het-cPA and het-cOR strains 136 Figure 3-24. Sequence comparison o f P 0 3 , P 0 4 , OD1 and G O l 138 Figure 3-25. Sequence comparison o f D E L 3 , H E T - c G R , H E T - c 0 R and H E T - c P A 140 Figure 3-26. Phenotype of transformants of allele del3 141 Figure 3-27. Average growth rate of allele del3 transformants 141 Figure 3-28. Sequence comparison o f P26 and PD1 144 Figure 3-29. Sequence comparison o f PD3 and O R 144 Figure 3-30. Secondary structure prediction for H E T - c specificity region 153 Figure 3-31. Proposed protein conformation in the variable domain for determining H E T - c specificity 157 Figure 3-32. Comparison of the predicted secondary structure of PD1 and H E T - c 0 R in the specificity region 158 Figure 4-1. Outline of het-cOR:GFP fusion construction 166 Figure 4-2. Outline of het-cOR:HA and het-cOR(PA) :HA fusion gene construction 169 Figure 4-3. Outline of het-cOR:HA fusion gene construction in pCB1004 vector 171 viii Figure 4-4. Summary o f predicted fusion gene products 176 Figure 4-5. Western blot analysis o f mouse anti-GFP immunoprecipitation samples with mouse anti-GFP antibody 182 Figure 4-6. Western blot analysis of mouse anti-GFP immunoprecipitation samples with polyclonal anti-HA antibody 183 Figure 4-7. Western blot analysis of polyclonal anti-HA immunoprecipitation samples with anti-HA antibody 184 Figure 4-8. Western blot analysis of polyclonal anti-HA immunoprecipitation samples with monoclonal anti-GFP antibody 185 Figure 4-9. The putative leucine rich haptad repeat region o f H E T - c polypeptides 190 Figure 4-10. Schematic model for stable H E T - c heterocomplex formation 194 Figure 5-1. General model of how het-c mediates vegetative incompatibility 199 ix List of Tables Table 2-1. Strains used for phylogenetic analysis 45 Table 2-2. Comparison of synonymous and nonsynonymous nucleotide substitutions per 100 sites in het-c polymorphic and non-polymorphic region 76 Table 3-1. List o f strains as transformation recipients 88 Table 3-2. List o f strains used for heterokaryon incompatibility tests 89 Table 3-3. Oligonucleotide primers for the construction of het-c alleles 96 Table 3-4. Specific primer sets and template for the 1 s t round P C R to generate specific alleles 97 Table 3-5. Phenotypes of the three classes o f het-c transformants 110 Table 3-6. Summary of the phenotypes of the introduction naturally occurring OR het-c - type chimeric constructs into N. crassa 115 Table 3-7. Average growth rate o f the transformants containing naturally occurring het-cOR - type chimeric constructs 116 Table 3-8. Transformantion of naturally occurring het-cGR - type chimric constructs into /V. crassa 118 Table 3-9. Average growth rate of transformants containing naturally occurring het-c chimric constructs 119 Table 3-10. Transformantion of naturally occurring het-cPA - type alleles into 7V. crassa 122 Table 3-11. Average rate of growth of transformants containing naturally occurring het-cPA chimeric constructs 123 Table 3-12. Summary of the phenotypes of the transformants of het-c alleles pdl,pd2, pd3 andpd4 129 Table 3-13. Summary o f phenotype of pol and po2 transformants 134 Table 3-14. Summary of phenotype of po3, po4, odl and gol transformants 139 Table 3-15. Phenotypes of co-transformants ofpol/po2,pdl, pd2 andpd3 145 Table 4-1. Summary o f the phenotypes of introducing fusion gene constructs into N. crassa 177 Acknowledgements I am very grateful to: Dr. Louise Glass for giving me the opportunity to complete my study in her laboratory and for her supervision, precious advice and support during the course o f this work. The members of my thesis committee, Dr. J im Kronstad, Dr. Sally Otto and Dr. Miche l Roberge for their advice on the research and the dissertation. In particular, Dr. Anthony Griffiths for providing me the space in his laboratory for the completion o f my research after the departure of Dr. Louise Glass; Dr. Jenneat Whitton for generously providing the phylogenetic analysis program and the computer. Dr. Carl Douglas, for the understanding, support and advice on my career choice. Rostam Namdari (Monka) for being there all the time, listening to all my frustrations as well as joys (Dollars!), for his caring, encouragement, and long-time friendship. Dorian Medd who came to my life with love at such a subtle phase o f my life M y family in China, for their love, their patience with and understanding of my never-ending student life. A l l the colleagues present and past in the Glass Lab and the Griffiths Lab, for their help, advice and many more.... Friends from Botany Department and Biotechnology Laboratory. Especially people from Tony Glass's lab, Kronstad's lab, Finlay's lab, Jeffrey's lab.. . . M y friends for sharing my frustrations and J O Y s with, Tania Thenu, Suman, Greg Lezee, Reza, Fatima, Fiona, Rebacca, Dr. Mary Berbee and Dr. Daniel Haydon for their help with phylogenetic analysis. Dr. Ken-ichiro Ishida for suggestions about the phylogentic analysis and critical reading of this part of the dessertation. Botany Department and Biotechnology Laboratory for the friendly environment and financial support. xi Dedications Dedicated to my forever-beloved parents, for their patience with and understanding of my never-ending student "life". Also to my dear siblings, in particular to my sister Rose (Yueji) and her family, for their support and taking my obligations towards our senior parents. Also, in the memory of my dear Mother. Donglan xii Chapter 1 General Introduction 1.1 SELF AND NON-SELF RECOGNITION Self and non-self recognition is a fundamental biological process that is essential for sexual reproduction, defense against pathogen invasion and maintenance o f individuality and integrity for an organism. Two apparently diametrically opposed mechanisms: self-incompatibility or non-self incompatibility, i . e., caused by identical or different gene products, may induce the recognition and subsequent discrimination reactions between cells or organisms. The self-incompatibility mechanism is active in the sexual reproduction process o f some hermaphroditic species, favoring outcrossing within the species (Esser and Blaich, 1973). The non-self incompatibility mechanism is active in somatic recognition between cells o f different individuals, favoring the integrity of the individual or species. The paradox of recognition of identity and difference applies to a variety o f systems. In 1968, Burnet first suggested that the ability to distinguish " s e l f and "non-se l f appeared initially in predatory protozoa, in which the amoeba has to discriminate between engulfed prey and itself (Burnet, 1968). Cell-cell recognition was also suggested to be associated with the emergence of sexuality (Monroy and Rosati, 1979). In prokaryotes, cell-cell recognition is best elucidated in the so-called "sexual events" in the bacteria Escherichia coli. The conjugation events between F + and F" bacterial strains are mediated by recognition of the cell surface glycoprotein encoded by the F "sex" plasmid (Willetts and Skurray, 1980). In eukaryotic cells, self and non-self recognition occurs during both sexual and asexual (somatic) growth, although different mechanisms are involved. 1 Identical gene products may cause self-incompatibility during sexual reproduction and different gene products may cause non-self incompatibility in somatic growth. These parallel phenomena are discussed in mammals, plants and ascomycete fungi. 1.1.1 Self and non-self recognition in mammals 1.1.1.1 Sexual recognition In mammals, the male and female individuals have specialized sex organs for production of different reproductive cells (gametes). Reproduction requires the initial recognition o f the female and male reproductive cells. The female reproductive cell, the egg, is surrounded by a glycoprotein layer that is responsible for mediating recognition of the male reproductive cell and thereafter fertilization (Beleil and Wassarman, 1980; Hanada and Chang, 1972). Genetically, however, sexual incompatibility does not restrict inbreeding. The male and female reproductive cells may carry identical genetic information (except for the male determination chromosome) in a random mating population. Promotion o f outbreeding is generally achieved by dioecism (for review, see Esser and Blaich, 1973). 1.1.1.2 Somatic incompatibility In mammals, the major histocompatibility complex encoded by an array o f Mhc loci controls somatic non-self recognition and subsequent activation o f defense mechanisms (for reviews, see Bjorkman, 1990; Kle in , 1980; 1986). M H C molecules were initially defined as the main target antigens in organ transplantation reactions. When organ grafts are exchanged between adult individuals, either of the same species {allografts) or o f different species (xenografts), they were usually rejected in response to the foreign antigens on the surface of 2 the grafted cells. These genetically "foreign antigens" or cell surface proteins were identified as major histocompatibility complex molecules ( M H C molecules). The major histocompatibility complex (MHC) is a multigene family encoding cell-surface glycoproteins that play a key role in immune response (Klein, 1986). The M H C glycoproteins and the T cell surface receptors (TcRs) are the key elements of specificity in the T cell response to foreign antigens. The M H C glycoproteins can be considered as antigen-presenting molecules. The antigenic peptide must be bound by a M H C glycoprotein to form a complex that can be recognized by the TcRs. The MHC encodes two categories of cell surface glycoproteins called class I and class U molecules, both of which are transmembrane heterodimers (reviewed in Bjorkman, 1990; Hughes and Yeager, 1998). Class I M H C molecules are expressed on the surface of all somatic cells and function to destroy virus-infected cells by presenting foreign-processed peptides to TcRs of cytotoxic T cells. The class U molecules are expressed on the surface of cells such as B cells, dendritic cells, macrophages, and epithelial cells. The class II molecules bind foreign peptides (foreign antigens) to form a ligand complex and present the complex to the TcRs of helper T cells to activate an immune response. The class II molecule is a heterodimer made of an a chain and a p chain, both of which are encoded in the class II region o f the M H C complex. The class I M H C molecules are non-covalently linked heterodimers, consisting of an a chain (or heavy chain) and a molecule called P2-microglobulin (P2m). The a chain is made up of three extracellular domains (designated as cti, ct2 and 013), a transmembrane region and a cytoplasmic domain. The P2m molecule only has a single domain extracellularly. The a chains are encoded within the M H C complex by the class I loci , o f which there are three in 3 humans, HLA-A, HLA-B, and HLA-C. The class I loci are polymorphic. The fcm molecule is encoded by a non-polymorphic locus outside the M H C complex. Both classes of M H C play an essential role in immune defense against foreign pathogens. However, there appears to be no specific mechanisms for distinguishing self-proteins from foreign antigens at the level of peptides binding to M H C molecules (Lorentz and Al len , 1988; 1989). The discrimination between self-proteins and non-self proteins occurs at the level of the TcRs that can only recognize foreign (non-self) peptides in the context of se l f -MHC molecules. When the cell is infected with a virus or other intracellular antigens, the viral peptides or other foreign peptides w i l l compete with endogenous self-peptides for binding to the M H C molecules on the surface o f the infected cell. Only when the TcRs of the T cells encounter the complex of se l f -MHC molecules and foreign peptides, an aggressive T cell response w i l l be stimulated. Both class I and class U M H C molecules appear to have similar peptide binding regions (PBR) in their extracellular domains (Bjorkman et al., 1987a, b; Brown et al., 1988, 1993). Highly polymorphic features were found in the P B R region o f both molecules that appear to enhance the capacity of each molecule to bind various foreign peptides (Klein, 1986; Fa lke ta l . , 1991). M H C molecules are expressed on the cells of all higher vertebrates. They were first found in mice and called H-2 antigens (histocompatibility-2 antigens, K le in , 1979). In humans they are called HLA antigens (human-leukocyte-associated antigens) because they were first demonstrated on leukocytes (for review, see Kle in , 1986). They have been identified in birds (Bourlet et al., 1988), amphibians (Flajnik et al., 1991) and fish (Hashimoto et al., 1990; Mi l l e r and Withler, 1996). Each individual has five or more loci 4 encoding M H C molecules. Many of the functional MHC loci are the most polymorphic loci known in higher vertebrates (Klein, 1979, 1986; K l e i n et al., 1993; Ono et al., 1993; Mi l l e r and Withler, 1996); that is, within a species there is a large number o f alleles at each locus and each allele is present at a relatively high frequency in the population. For example, more than 149 alleles have been identified at the human MHC class I locus HLA-B (Klein, 1986; Parhem and Ohta, 1996); more than 126 DRB alleles have been identified at the human MHC class IIDRB locus (Yuhki and O'Brien, 1997). Despite the enormous diversity of M H C molecules, polymorphism and allelic lineages at the functional class I and class II MHC loci are very stable, persistent and shared between different species. These evolutionary characteristics of MHC loci are believed to be maintained by balancing selection (Doherty and Zinkernagel, 1975; Hughes and Ne i , 1988, 1989). Supporting evidence for this conclusion includes: (1) a large number of alleles occur at functional MHC loci (reviewed in Kle in , 1986); (2) allele frequencies are distributed more or less evenly in a population (Hedrick and Thomason, 1983); (3) Trans-specific polymorphism was found at both class I and class II loci (i . e. certain alleles o f one species are generally more similar to certain alleles of another species than they are to other alleles of the same species (Klein, 1980; Figueroa et al., 1988; Lawlor et al., 1988; Mayer et al., 1988; McConnel l et al., 1988)); (4) the rate of nonsynonymous substitutions per site at the variable P B R is higher than that o f synonymous substitutions per site (Hughes and Ne i , 1988; 1989). 1.1.2 Sexual incompatibility in plants - the S locus In many flowering plant species, sexual reproduction is mediated by the gametophytic or sporophytic self-incompatibility locus, the S locus (for reviews, see Nasrallah and 5 Nasrallah, 1986; Haring et al., 1990; Charlesworth, 1994; 1995). Self-incompatibility is a mechanism by which many flowering plants prevent self-fertilization and promote outbreeding. B y contrast to the M H C complex, it is a mechanism for self- recognition that results in rejection of self-pollen by the female somatic tissues. In certain species, the specificity of the self-incompatibility interaction has been shown to be controlled by the S locus (for review, see Charlesworth, 1994; 1995). The two self-incompatibility systems differ in the phenotype expressed by the pollen grain during pollination. In gametophytic self-incompatibility, represented in the Solanaceae (Nicotiana, Petunia, and Solanum species), the single S-allele o f the haploid pollen grain determines self-incompatibility (reviewed by Kao and McCubbin , 1996). Fertilization is prevented i f the S allele carried by the pollen is identical to one o f the S alleles of the recipient plant. In the sporophytic system, represented in the Brassicaceae and exemplified by the genus Brassica, the interaction o f the two S alleles that are borne by the two diploid parent plants (sporophyte) determines the self-incompatibility phenotype (Goodwillie, 1997). The cellular mechanism of recognition of both types o f self-incompatibility is not well understood (for reviews, see Clarke and Newbigin, 1993; Kao and McCubbin , 1996; Nasrallah, 1997). The S loci exhibit allelic polymorphism at high levels in both systems (reviewed by Charlesworth, 1995). The S locus o f the gametophytic self-incompatibility system encodes a pistil-specific protein, the S protein, which functions in the recognition and rejection o f pollen bearing the same allele (Haring et al., 1990). In members of the Solanaceae, the S locus encodes glycoproteins with ribonuclease (RNase) activity (McClure et al., 1989). The role of RNase activity in self-incompatibility is unclear although several models have been proposed (Kao and McCubbin , 1996; Hiscock et al., 1998). 6 Sporophytic self-incompatibility systems have been described in a number of species and extensively studied in Brassicaceae. In Brassica species that exhibit self-incompatibility, two tightly linked genes, SLG and SRK, at the S locus are involved in the recognition reaction with self and non-self pollen (for review, see Trick and Heizmann, 1993; Hatakeyama et al., 1998). The SLG (S locus glycoprotein) gene encodes a highly polymorphic secreted glycoprotein that localizes primarily to the cell wal l o f epidermal cells of the stigma (Nasrallah et al., 1985, 1987; for review, see Hiscock et al., 1998), whereas the SRK (S locus receptor kinase) gene encodes a transmembrane serine/threonine protein kinase with an extracellular domain (S domain) that spans the plasma membrane (Stein et al., 1996). Several lines of evidence suggest that both of these genes are required for the recognition reaction of self-incompatibility (see reviews, Nasrallah and Nasrallah, 1986; Hiscock et al., 1998; Nasrallah, 1997). Current models predict a central role for S R K in the initiation of the self-rejection response, yet the molecular and biochemical basis of the signaling pathway is unknown (Hiscock et al., 1998). The gametophytic incompatibility system has been most extensively studied in the Solanaceae. S alleles have also been sequenced from species in the Papaveraceae, Onagraceae and Gramineaceae (for reviews, see Charlesworth, 1995; Kao and McCubbin , 1996). The S alleles of the sporophytic incompatibility system have also been identified in species of other families (the Primulaceae and the Asteraceae) besides those in the Brassicaceae (reviewed by Charlesworth, 1995). A high degree of D N A sequence variability was found among S alleles, even between alleles from the same species. A s with the MHC, S loci exhibit several features of loci under balancing selection: 1) A large number of alleles occur in a population. In the Solanaceae, populations commonly harbor as many as 30 to 50 alleles (Lane and Lawrence, 7 1993; Sakamoto et al., 1998). 2) S allele polymorphisms are shared among species and arose prior to speciation (Ioerger et al., 1990). That is, alleles at the S locus in the Solanaceae show "trans-specific" mode o f evolution (Ioerger, et al., 1990; Richman et a l , 1995). 3) Al le le polymorphisms have persisted for an evolutionarily significant period of time (Hinata et al., 1995). Polymorphisms observed in the S alleles in members of the Solanaceae have persisted for at least 36 mil l ion years (Clarke and Kao, 1991; Richman et al., 1996). 4) A n excess of nonsynonymous nucleotide substitutions over synonymous nucleotide substitutions was observed at shared polymorphic sites (Clark and Kao, 1991; Hinata et al., 1995). A high nonsynonymous nucleotide substitution rate was observed in the polymorphic region of both SLG and SRK loci (Hinata et al., 1995) and at the shared polymorphic sites among S alleles in the Solanaceae (Clarke and Kao, 1991). 1.1.3 Self and non-self recognition in ascomycete fungi In fungi, non-self recognition events occur in both sexual and vegetative reproduction stages. Sexual reproduction in outcrossing species is governed by mating-type genes that determine the pattern of mating among individual strains. Recognition mediated by these genes allows sexual development. The mating recognition system mostly restricts inbreeding and promotes outbreeding and thereby maintains genetic variability within a population. Filamentous fungi also possess a somatic recognition system to restrict hyphal fusion between genetically non-identical individuals. Such a system is referred as vegetative or heterokaryon incompatibility and is genetically regulated by het (for /jererokaryon) loci. 8 In filamentous ascomycetes, mating recognition and vegetative incompatibility in general do not interfere with each other. However, in some species, such as Neurospora crassa or Ascobolus immersus, the mating type genes also function as het genes that regulate vegetative incompatibility during the asexual reproductive phase. 1.1.3.1 Mating-type and mating recognition in ascomycetes In fungi, the mating-type locus (mat) controls mating recognition. The mating type system in fungi was first identified in the zygomycete Rhizopus (Blakeslee, 1904), in which successful mating only occurs between two sexually compatible strains. Isolates of this fungus are morphologically indistinguishable, but sexually divided into two compatible mating groups designated as "+" and Subsequently, mating types were discovered in ascomycetes (Edgerton, 1914) and basidiomycetes (Bauch, 1927). Several mating strategies exist in ascomycetes, including heterothallism, homothallism and pseudohomothallism (reviewed in Nelson, 1996). Heterothallic species (such as N. crassa, N. sitophila, N. discreta, and Cochliobolus heterostrophus) are self-sterile. Mating requires strains of opposite mating-types to come together during the sexual process. Homothallic species (such as N. pannonica, N. dodgei) are self-fertile, in which a single haploid nucleus carries all the information necessary for the sexual process. Pseudohomothallic species superficially resemble homothallic species in that they are also self-fertile. Unlike homothallic species, pseudohomothallic species (such as N. tetrasperma, Podospora anserina) have two opposite mating-type nuclei that are compartmentalized into a single ascospore. Thus, pseudohomothallic species do not exhibit mating-type associated 9 vegetative incompatibility; stable heterokaryons are formed by nuclei carrying opposite mating-type genes. The mating system in heterothallic and pseudohomothallic ascomycetes is bipolar - a one-locus, two-"allele" mating system (for reviews, see Glass and Kuldau, 1992). Mating recognition is mediated at two steps: one involves recognition that controls initial plasmogamy; the other involves recognition of opposite mating-type nuclei for post-fertilization development. In the unicellular ascomycetes, studies on mating recognition have been conducted in Saccharomyces cerevisiae and Schizosaccharomyces pombe (for review, see Herskowitz, 1989). In filamentous ascomycetes, studies on mating recognition have been conducted mainly in N. crassa, P. anserina, and C. heterostrophus (for reviews, see Glass and Kuldau, 1992; Coppin et al., 1997; Kronstad and Staben, 1997). The molecular mechanisms o f mating recognition in representative species w i l l be described in Section 1.2.1 and 1.2.2. 1.1.3.2 Vegetative incompatibility in filamentous ascomycetes In filamentous fimgi, somatic cell-cell recognition regulates the success o f hyphal fusion and heterokaryon formation during vegetative growth phase. When vegetative compatibility permits heterokaryon formation, heterokaryotic growth occurs. In many fungal species, however, vegetative incompatibility is observed by the occurrence of a barrage between incompatible mycelia, which is a macroscopic line o f numerous degenerated and dead hyphal compartments that underwent anastomoses (Beguret et al., 1994; Esser and Blaich, 1994; Figure 1-1). In the laboratory, vegetative incompatibility can be detected directly by forced heterokaryon formation between two auxotrophic strains. Hetrokaryons 10 formed between two strains that have alternative alleles at a het locus are inhibited in their growth, displaying hyphal compartmentation and subsequent destruction by a lytic process (for reviews, see Glass and Kuldau, 1992; Leslie, 1993; and Begueret et al, 1994). Figure 1-1. Barrage zone between fungal strains that are vegetatively incompatible, a, Barrage formation between vegetatively incompatible P. anserina isolates. A contact between two incompatible strains is shown by the solid black arrow. The other arrow shows a contact between compatible strains (Adapted from Begueret et al., 1994). b, Cross section o f a log colonized by different isolates of basidiomycetes (Adapted from Esser and Blaich, 1994). Border region between incompatible mycelial is shown by dark lines that result from regeneration of apical hyphae after the death of incompatible hyphal compartments. Understanding vegetative incompatibility and its genetic control mainly comes from studies in the filamentous ascomycetes /V. crassa and P. anserina, both of which are in the Sordariaceae (for reviews, see Glass and Kuldau, 1992; Leslie, 1993; Begueret et al., 1994). In a given species, numerous het loci occur, e. g. at least ten het loci have been identified in N. crassa and nine have been identified in P. anserina (for reviews, see Begueret et al., 11 1994). Al le l i c differences at any of these het loci between two individuals are sufficient to cause a vegetative incompatibility reaction upon hyphal anatomosis. Molecular and cellular aspects of vegetative incompatibility in these two model species w i l l be discussed in detail in Section 1.3. Vegetative incompatibility is widespread in fungi. Its biological significance in fungal populations is however, still unclear. It is not known whether vegetative incompatibility and vertebrate graft rejection phenomena arose by convergent evolution (Esser and Blaich, 1973) in each group or whether vegetative incompatibility is a primitive trait o f allorecognition or immune response (Lane, 1981). Vegetative incompatibility is believed to prevent transmission of conventional parasites such as mycoviruses and detrimental cytoplasmic elements, which are spread via hyphal fusion (Caten, 1972; Anagnostakis, 1983; Debets et al., 1994; Nauta and Hoekstra, 1994; van Diepeningen et al., 1997). Advantages such as preventing nuclear invasion or genomic replacement after anastomosis has also been suggested (Todd and Rayner, 1980; Rayner et al., 1984; Rizzo and Harrington, 1992; Rizzo and May, 1994; for review, see Worrall , 1997). Vegetative incompatibility may also limit outbreeding and genetic polymorphism within the species, especially in pseudohomothallic species. In particular, it limits the chance of somatic genetic recombination that is important in imperfect fungi (e. g. Aspergillus niger and Fusarium oxysporum) that have lost their sexuality (Caten, 1971). 12 1.2 MOLECULAR ASPECTS OF NON-SELF RECOGNITION IN ASCOMYCETES Understanding the molecular mechanisms of mating recognition in fungi has been explored with a combination of genetic and biochemical studies (for reviews, see Kahmann and Bolker, 1996; Kronstad and Staben, 1997). The molecular and biochemical basis of vegetative incompatibility is not as well characterized. Nonetheless, the understanding of mating recognition may provide concepts for how vegetative incompatibility is accomplished. In fungi, non-self recognition in mating is accomplished through two distinct molecular mechanisms. One is via a pheromone signaling pathway to choose a mating partner. The other involves heterodimerization o f proteins to gain new regulatory functions favoring nuclear recognition and proliferation after mating. These mechanisms have precedents in both yeast and filamentous ascomycete mating systems. 1.2.1 Mating recognition in Yeast The best characterized mating recognition system in fungi is that o f the yeast S. cerevisiae. Mating recognition in this organism relies on a complex signal transduction pathway via mating-type specific pheromone receptors (for review, see Johnson, 1995). Cells of one mating-type respond only to pheromone produced by cells o f the opposite mating-type. The laboratory maintained heterothallic budding yeast S. cerevisiae has two mating-types, a and a, encoded by the MAriocus. Three cell types with different properties exist in its life cycle: the haploid a cell and a cell and the diploid a/a cell. S. cerevisiae does not have 13 specialized reproductive structures and is competent for mating during vegetative growth without the requirement for nutrient depletion. Mating occurs only between haploid a cells and a cells; a/a cells can not mate (for review see Borkovich, 1996). The mat a locus encodes M A T c c l and MAToc2; the mat a locus encodes M A T a l (for review, see Kues and Casselton, 1992). In haploid cells, M A T a l activates a-cell specific genes, i . e. pheromones and pheromone receptors; MAToc2 represses the expression of a-specific haploid genes. The M A T a l is not required for the expression of any a-specific haploid genes (Fields, 1990; for review, see Kues and Casselton, 1992). The haploid a cells and a cells secrete cell-specific peptide pheromones, the a-pheromone and the a - pheromone, respectively. Each haploid cell posseses a seven transmembrane pheromone receptor on the cell membrane for binding pheromones produced by the opposite mating type cells (Burkholder and Hartwell, 1985; Hagen et al., 1986; for review, see Cross, 1988). For instance, the pheromone receptor Ste2 protein on the membrane of the a cells binds a pheromone, while the Ste3 protein on the surface of the a cells is the receptor for a pheromone. The pheromone-receptor ligand induces expression of a set of genes in the a cells and a cells v ia a G-protein and M A P kinase signaling pathway, resulting in cell differentiation and formation of a protrusion, or "schmoo". Such a structure allows physical contact and thus conjugation and nuclear fusion between the a cell and a cell to form a diploid a/a cell (Figure 1-2). In diploid a/a cells, the two homeodomain proteins M A T a l and M A T a 2 form an a l - a 2 heterodimer that acts as a transcriptional repressor of haploid specific genes such as R M E 1 (repression of meiosis) and allows meiosis and sporulation to ensue under appropriate nutritional conditions (Dranginis, 1990; Covi tzet al., 1991). 14 r RECEPTOR SIGNALLING ICX AGGLUTMINS CDC24 (SITE SELECTION: OtRECTEO SHMOOT] RECOVERY: CYCLING ZYGOTE . CELL FUSION: HETEROKARYON KAR1.2.3 TU88 C0C4.28.34J7 (CVTO. MTS: SPBT) (NUCLEAR M00IF.7) NUCLEAR FUSION: G1 ZYGOTE Figure 1-2. The mating process in Saccharomyces cerevisiae. The events that occur during conjugation are shown. Cells are shown as starting the mating process in the G I phase of the cell cycle before spindle-pole body duplication. G I arrest mediated by the presence of a- and a-pheromones w i l l result in the arrest of cells at this point prior to mating. (Adapted from Cross etal., 1988). 15 1.2.2 Mating recognition in filamentous ascomycetes Mating recognition in filamentous ascomycetes has features in common with yeast v ia mating type-specific pheromone response signaling pathways controlled by mating-type loci (for review, see Kronstad and Staben, 1997). In the following sections, an overview w i l l focus on the well-characterized mating systems from the two representative filamentous ascomycetes, TV. crassa and P. anserina. 1.2.2.1 Life cycle of filamentous ascomycetes The principal events of the life cycle are similar between 7V. crassa and P. anserina except that asci in the pseudohomothallic species, P. anserina, contain four ascospores in which two nuclei of opposite mating-types are enclosed. A s an example o f filamentous ascomycetes, the life cycle of TV. crassa is presented in Figure 1-3. It consists of vegetative and sexual growth phases. Asexual (or vegetative) reproduction generally occurs by either mycelial propagation or by the production of numerous vegetative spores, known as conidia (Alexopoulos, 1962). Under nutrient starvation, particularly nitrogen depletion (Westergaard and Mitchel l , 1947), N. crassa switches from vegetative growth to sexual development. The sexual cycle begins with the differentiation of female reproductive structures, protoperithecia, which are composed of a spherical mass of tightly coiled hyphae. A specialized hypha, called trichogyne, grows directionally from the protoperithecium towards a male cell (typically hyphae or asexual spores) of the opposite mating-type and fuses with the male fertilizing cell. This directional growth is believed to be mediated by mating-type (A and a) pheromones (Bistis, 1981; 1983). Nuclei from the male are transported into the ascogonium after cell 16 fusion. The proliferation of haploid nuclei of both mating-types results in the formation of dikaryotic ascogenous hyphae, followed subsequently by nuclear fusion between opposite mating-type nuclei. The diploid nucleus immediately undergoes meiosis and eight haploid ascospores are formed in a sac-like structure called an ascus. Figure 1-3. Life cycle o f TV. crassa. (Adopted from Nelson, 1996). 17 1.2.2.2 The mating-type locus The mating-type locus in filamentous ascomycetes contains dissimilar D N A sequences that do not seem to have an allelic relationship, for which the termed idiomorph was adopted (Metzenberg and Glass, 1990). Idiomorphic structure is common to most known fungal mating-type genes, including those from the unicellular ascomycetes S. cerevisiae and S. pombe. Mating-type idiomorphs of N. crassa are termed mat A and mat a (Metzenberg and Glass, 1990). The mat A idiomorph contains three mating-type genes, matA-1, matA-2 and matA-3, among which only matA-1 is essential for mating recognition (Ferreira et al., 1996). The mat a idiomorph contains only one transcription unit mat a-1, which is essential for mating recognition (Chang and Staben, 1994). The mating idiomorphs of P. anserina are similar to those of N. crassa, but are designated as mat+ and mat- (for review, see Glass and Kuldau, 1992). The mat+ idiomorph has a single gene, FPR1, that is homologous to mat a-1 in N. crassa. The mat- idiomorph contains three genes, FMR1, SMR1 and SMR2, with similarity to matA-1, matA-2 and matA-3, respectively (Debuchy and Coppin, 1992; Debuchy et al., 1993). Two genes, FPR1 and FMR1, mediate mating recognition. 1.2.2.3 Pheromone response pathway Similar to S. cerevisiae, it is believed that mating-type in filamentous ascomycetes functions to control recognition mechanisms leading to fertilization through a pheromone response pathway. In N. crassa, males of each mating-type secrete a pheromone to which females of the opposite mating-type respond by orienting growth of their trichogynes (Bistis, 1983). Mating-type mutants ofN. crassa strains do not orient hyphal growth towards 18 pheromone source or produce pheromone (Bistis, 1996), suggesting that mating-type specific pheromones and their receptors are likely to be regulated by mating-type gene products. 1.2.2.4 H i g h - M o b i l i t y - G r o u p ( H M G ) and a l domain proteins A D N A binding motif, the high-mobility-group ( H M G ) domain was found in the M A T proteins o f N. crassa ( M A T a-1 and M A T A-3) , and P. anserina (FPR1 and SMR2) (reviewed by Coppin et al., 1997). A subregion of the M A T A-1 (N. crassa) and F M R 1 (P. anserina) polypeptide has similarity to the protein encoded by the matal gene of S. cerevisiae. It has been proposed that M A T a-1 in N. crassa controls mating via the D N A binding activity of its H M G domain (Philley and Staben, 1994). Whether M A T a-1 and M A T A-1 dimerize, as in the case of S. cerevisiae ct2-al, to confer a novel cell-type is unknown. In P. anserina, the two genes FPR1 (homolog of mat a-1 in 7Y. crassa) and FMR1 (homolog of mat A-1 in TV. crassa) are responsible for mating specificity. Yeast two-hybrid data failed to support an interaction o f FPR1 and F M R 1 , suggesting that some other mechanisms may exist for the regulation o f post-fertilization functions (Coppin et al., 1997). However, yeast two-hybrid analysis showed an interaction between M A T A-1 and M A T a-1 in TV. crassa (C. Staben, personal communication). 19 1.3 VEGETATIVE INCOMPATIBILITY IN FILAMENTOUS ASCOMYCETES Vegetative incompatibility has been described in many filamentous ascomycetes (reviewed by Esser and Blaich, 1994; Leslie, 1993; Begueret et al., 1994). The genetic basis of incompatibility has been elucidated in several filamentous ascomycetes such as Aspergillus nidulans (Grindle, 1963), Cryphonectria parasitica (Anagnostakis, 1977), N. crassa (Gamjobst, 1953, 1955; Perkins, 1988) and P. anserina (Bernet, 1967). However, studies conducted in most species are limited to population analysis due to the lack of genetic and molecular tools. Understanding the cellular, biochemical and molecular basis of vegetative incompatibility has mainly been conducted in two species of filamentous ascomycetes, the pseudohomothallic species, P. anserina, and the heterothallic species, N. crassa. Genes involved in vegetative incompatibility have been cloned and molecularly characterized from both species (for reviews, see Begueret et al., 1994; Glass and Kuldau, 1992; Saupe et al., 1996; Saupe and Glass, 1997). However, many aspects of the cellular and biochemical bases o f vegetative incompatibility remain unclear. 1.3.1 Vegetative incompatibility in Podospora anserina In P. anserina, genes present at het loci can be involved in both allelic and non-allelic interactions (for review see Begueret et al., 1994). Nine het genes have been identified. Three het loci , het-s (Turcq et al., 1990), het-c (Saupe et al, 1994) and het-e (Saupe et al., 1995) have been molecularly characterized. The het-c locus o f P. anserina has no similarity with the het-c locus in TV. crassa that w i l l be described in section 1.3.2. Among the nine known het loci , het-c, het-d, het-e, het-r and het-v are involved in non-allelic incompatibility; non-allelic 20 genetic interactions between het-c/het-e, het-c/het-d, and het-r/het-v have been characterized (Bernet, 1967; 1992). The het-s locus mediates allelic incompatibility and encodes two mutually incompatible alleles, het-s and het-S. Hyphal fusion between wi ld type P. anserina het-s and het-S strains triggers a vegetative incompatibility response and cell death (for review, see Esser and Blaich, 1994). Disruption of the het-s locus does not affect cell viability and fertility (Turcq et al., 1991). The het-s and het-S alleles encode similar peptides of 289 amino acids, differing by 14 amino acids. Chimeric construction and site-specific mutagenesis showed that a single amino acid difference between the two polypeptides was sufficient to elicit vegetative incompatibility (Deleu et al., 1993). How this difference in amino acid composition mediates vegetative incompatibility is unclear. A neutral allele, het-s*, was also identified in w i ld type isolates (Bernet, 1967). Strains containing the het-s* allele are compatible with both het-s and het-S strains. A het-s* strain can switch to het-s specificity spontaneously at a low frequency or after fusion with the hyphae of a het-s strain (Deleu et al., 1993). Expression of the HET-s protein was found at the same level in het-s and het-s* strains, which led to the conclusion that a post-translational modification o f the HET-s protein is necessary for the phenotypic differences between het-s and het-s* strains (Begueret et al., 1997). Several lines of evidence indicate that the HET-s protein shares features with the prion protein (Begueret et al., 1997; personal communication). Firstly, both are able to form homodimers. Secondly, like the prion protein, the HET-s can exist in two forms which differ in the sensitivity to proteolytic enzymes. A n d thirdly, overexpression of HET-s can enhance the switch of het-s* strain to het-s strains. 21 The het-c and het-e loci are multiallelic and are involved in non-allelic incompatibility. Genes have been cloned from these two loci. The het-e locus contains an open reading frame encoding a polypeptide o f 1356 amino acids with two interesting sequence motifs. The predicted H E T - E protein contains functional domains similar to those present in a and P subunits of heterotrimeric G proteins: the GTP-binding motif at the N -terminal and Gp homologous domain (or the W D 4 0 repeated domain) at the C-terminal (Saupe et al., 1995a). Mutational analysis and in vitro binding assays indicated that both the G T P binding domain and the W D 4 0 repeats were required for vegetative incompatibility. It was suggested that H E T - E might be involved in signal transduction mediating vegetative incompatibility (Espagne et al., 1997). Four alleles were identified at the het-c locus in wi ld-type strains. The four het-c alleles encode similar polypeptides of 208 amino acids with similarity to glycolipid transfer proteins (Saupe et al., 1994; 1995b). Polypeptides encoded by the different het-c alleles contain 16 polymorphic positions and a single amino acid difference was found sufficient to modify allele specificity (Saupe et al, 1995b). It was suggested that selection pressure maintained the high degree o f polymorphism at the het-c locus based on the observation of excess nonsynonymous nucleotide substitutions at polymorphic sites among the four het-c alleles (Saupe et al, 1995b). Disruption of het-c locus dramatically affects ascospore formation, suggesting that het-c is essential in the life cycle of P. anserina. However, how interaction between H E T - E and H E T - C mediates vegetative incompatibility is unclear. Studies on biochemical mechanisms mediating vegetative incompatibility in P. anserina are under way by the isolation of genes (idi) induced upon vegetative incompatibility and suppressor mutations (or modifier mutations, mod mutations). Several idi 22 (idi-1, 2, 3) and mod (mod-Al, mod-D and mod-E) genes have been molecularly characterized. The putative DDI proteins are small proteins with signal peptides and were proposed to be involved in regulating different steps of the incompatibility reaction (Bourges et al., 1998). Most of the mod mutations affect both protoperithecial development and vegetative incompatibility, suggesting that the incompatibility reaction in P. anserina may be related to the regulation of differentiation. It has been proposed that het genes may control some differentiation steps in the life cycle of P. anserina, such as the transition from the quiescent to the proliferation state or the formation o f female organs (Bernet, 1992). The mod-Al mutation has no effect on allelic vegetative incompatibility interactions but suppresses incompatibility resulting from all three non-allelic systems (Bernet et al., 1973). The mod-Al mutation restores the growth of incompatible strains but does not suppress the cell lytic reaction (Bernet et al., 1973). The putative M O D - A polypeptide contains a proline-rich domain with similarity to SH3-binding motifs (Barreau et al., 1998). It was suggested that M O D - A is involved in differentiation and functions as key regulator of growth arrest associated with vegetative incompatibility (Barreau et al., 1998). The suppressor mutation mod-Bl does not modify the phenotype o f incompatibility of the non-allelic system, but the presence of both mod-Al and mod-Bl mutations suppresses the growth arrest and cell lytic reactions of non-allelic systems. The mod-Cl mutation specifically suppresses incompatibility in the het-R/het-Vstrains. The predicted M O D - D polypeptide is a Gcc subunit o f a heterotrimeric G protein that is involved in the c A M P signal transduction pathway. The mod-D mutant strains present developmental defects and partial suppression of vegetative incompatibility (Loubradou et al., 1999). A gene coding for an adenylate cyclase, PaAC, was also identified as a partial suppressor o f a mod-D mutant allele (Loubradou et al., 23 1996). The mod-E mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. The putative M O D - E is a member o f the HSP90 family, a possible component o f a signaling pathway involved in both vegetative incompatibility and development (Loubradou et al., 1997). Genetic and mutational studies suggested that the vegetative incompatibility response was triggered by the co-expression of incompatible het genes (for review, see Begueret et al., 1994). Based on these studies, a "poison heteromeric complex" model of het gene products has been proposed for mediating vegetative incompatibility in P. anserina (Begueret et al., 1994). It was proposed that the heteromeric complex formed by incompatible het gene products might have direct lethal effects on the cell, perhaps by disrupting gene regulation related to P. anserina development (Bourges et al., 1998). The molecular characterization of modifier genes that mediate incompatible interactions and are involved in differentiation may help explain the link between incompatibility and developmental regulation. However, no direct evidence is available that supports the hypothesis o f protein heteromeric complex formation during vegetative incompatibility. 1.3.2 Vegetative incompatibility in /V. crassa Studies of natural populations of TV. crassa found that no two isolates from the same geographical area were able to form a stable heterokaryon (Mylyk, 1976). Genetic determinants that limit the formation of stable heterokaryons have been wel l studied in N. crassa (Mylyk, 1975; Perkins, 1975). A t least ten het loci , including the mating type locus have been identified that regulate the capacity to form stable heterokaryons between different isolates (Mylyk, 1975; Perkins, 1975; 1988). The mating-type locus differs from other het 24 loci because it also regulates entry into sexual reproduction (Beadle and Coonradt, 1944; Sansome, 1946). Morphological features and cellular responses associated with vegetative incompatibility have been described (Perkins, 1988; Jacobson et al., 1998). Two het loci, mat and het-c, have been genetically and molecularly characterized. The het-6 locus was cloned recently (Smith et al., in press). Unlike P. anserina, non-allelic interactions between het loci have not been described in N. crassa, although they may occur at het-6 (Smith et al., in press). 1.3.2.1 Neurospora crassa, the model organism Neurospora is blessed with well-known advantages for genetic research. It is haploid with a relatively small genome. It has a short generation time and a fast growth rate. It grows on simple, defined media and can be preserved. Large numbers o f sexually produced progeny can be recovered for mapping of genes. Among Neurospora species, N. crassa was chosen as a model to study vegetative incompatibility for several reasons. A l l ten het loci have been well mapped genetically (Perkins, 1988). N. crassa translocation strains are available that allow the construction of partial diploids of particular het loci that may be either heterozygous or homozygous (Perkins, 1975). There are a large number o f laboratory-maintained isogenic strains available for heterokaryon tests. Gene mutations can be easily achieved by a unique process termed RIP (Repeated Induced Point mutation) that allows directional mutagenesis to specific D N A sequences (Selker et al., 1987; Cambareri et al., 1989). 25 1.3.2.2 Cellular response to vegetative incompatibility The cellular and biochemical mechanisms behind vegetative incompatibility remain largely unknown although morphological features have been described. One characteristic of vegetative incompatibility is hyphal compartmentation and lytic death (Figure 1-4). A description o f hyphal compartmentation and death during vegetative incompatibility has been provided by recent ultrastructural studies of incompatible partial diploids (Jacobson et al, 1998). N o apparent differences were found between hyphal compartmentation and death resulting from incompatible het-c, het-6, or mat partial diploids or heterokaryons (Jacobson et al., 1998; Glass, personal communication), suggesting that each het gene may initiate a common hyphal death pathway (Jacobson et al, 1998). Ultrastructural changes were obvious in incompatible partial diploid cells and transformed cells as compared with wild-type cells, i . e. hyphal compartmentation by septal plug formation, shrinkage o f cell membrane, degradation o f cell organelles, cytoplasm vacuolization and loss o f nuclear contents. Incompatible hyphae commonly show an "invasion" event in the death process; dying hyphae were "invaded" by adjacent healthy hyphae, which may subsequently undergo hyphal compartmentation and death (Figure 1-5). Ultrastructural features of incompatible hyphae exhibit similarities with programmed cell death (PCD) in animal and plant cells (Jacobson et al., 1998; Fraser and Evan, 1996; Havel and Durzan, 1996). Programmed cell death can be defined as an orderly process where the dying cell actively participates in its own death (Columbano, 1995; Fraser and Evan, 1996). It is in contrast to necrosis or degenerative death caused by the external environment (Columbano, 1995). Programmed cell death is morphologically characterized by "blebbing" o f the plasma membrane, formation of apoptotic bodies and chromatin condensation, and 26 biochemically characterized by fragmentation of nuclear D N A at internucleosomal sites (Cohen et al., 1994). Nuclear D N A degradation was detected by the T U N E L (Terminal U T P Nick End Labeling) assay in het-c incompatible transformants, suggesting that vegetative incompatibility might have some biochemical similarities to programmed cell death (Marek et al., 1999). However, whether the hyphal death triggered by vegetative incompatibility shares common genetic pathways with programmed cell death in animals or plants remains to be determined. Figure 1-4. Hyphal compartments in het-c incompatible heterokaryons. The dead and dying hyphae were stained within the vital stain, Evan's blue (arrows). 2 7 Figure 1-5. Ultrastructural characteristics of wild-type and incompatible hyphae (provided by Dr. Glass), a, wi ld type hyphae. b, hyphae of mating type mat A /mat a partial diploid, c, hyphae of incompatible het-c transformants by introduction of het-cOR into a het-cPA strain. Black arrows show the dying and dead cells. White arrows indicate septal plugs in dying and dead cells. 1.3.2.3 Mating-type associated vegetative incompatibil i ty in N. crassa The mating-type locus has two functions; it controls not only mating but also vegetative incompatibility. Fusion of reproductive structures from strains of opposite mating-types (mat A and mat a) initiates sexual reproduction. Hyphal fusion between mat A and mat a strains elicits an incompatibility reaction that results in the death of fused hyphal compartments (Beadle and Coonradt, 1944; Garnjobst, 1953; Gross, 1952; Staben and Yanofsky, 1990); only mat A-1 and mat a-1 are required for vegetative incompatibility (Glass et al., 1990; Staben and Yanofsky, 1990; Ferreira et al., 1996). 28 The mat A-1 geneencodes a 293 amino acid polypeptide with a region o f homology to the M A T a l mating-type polypeptide of S. cerevisiae (Glass et al., 1990). Functional dissection of the M A T A-1 polypeptide showed that a region of 99 amino acids at the N -terminal was required for female fertility and that a region o f 111 amino acids at the N -terminal (including the above 99 amino acids) was sufficient for conferring vegetative incompatibility, while male fertility required amino acids from position 1 to 227 (Saupe et al., 1996a). The mat a encodes a 382 amino acid polypeptide containing specific regions essential for mating and vegetative incompatibility. Functional dissection of the M A T a-1 polypeptide showed that the H M G domain was required for D N A binding and mating but dispensable for vegetative incompatibility (Philley and Staben, 1994). In contrast, the C-terminal acidic region was found necessary for mediating vegetative incompatibility but non-essential for mating. It has been proposed that the mat a gene controls vegetative incompatibility via interaction of the C-terminal part with another factor, while the H M G domain controls mating via its D N A binding activity (Philley and Staben, 1994). Mating-type associated vegetative incompatibility must be somehow suppressed during sexual reproduction to allow the proliferation of opposite mating-type nuclei in ascogenous hyphae. A recessive mutation unlinked to the mating-type locus, tol (for tolerant) was found to suppress mating-type associated vegetative incompatibility without affecting mating ability (Newmeyer, 1970). The tol locus encodes a 1011 amino acid polypeptide containing an amphipathic cc-helix and a leucine-rich repeat domain (Shiu and Glass, 1999). H o w M A T A - 1 , M A T a-1 and T O L mediate mating-type associated incompatibility during vegetative growth is unclear. 29 1.4 HET-C M E D I A T E D V E G E T A T I V E I N C O M P A T I B I L I T Y I N N. CRASSA The het-c locus was originally identified in forced heterokaryons (Garnjobst, 1953; Garnjobst and Wilson, 1956) and then further characterized by using translocation strains generating partial duplications of the het-c locus (Perkins, 1975). Incompatible heterokaryons or partial diploids heterozygous for het-c display a slow-growing, curly, flat, aconidiating phenotype (Perkins, 1988; Figure 1-6). a b Figure 1-6. Morphology of compatible and incompatible het-c heterokaryons. a, compatible het-c heterokaryons formed between two auxotrophic het-cPA strains, b. Incompatible heterokaryons formed between two auxotrophic strains that are het-cPA and het-cOR at the het-c locus, respectively. The het-c locus is located on the left arm of LGII , ~ 1 map unit centromere distal to pyr-4. The het-c locus encodes three distinct and mutually incompatible alleles, het-c0R, het-cPA and het-cGR (Saupe and Glass, 1997). The het-cOR allele encodes a 966 amino acid 30 polypeptide, with a signal peptide in the N-terminal, an amphipathic alpha-helical region and OR a glycine-rich domain at the carboxy-terminal (Figure 1-7). Introduction of a mutated het-c allele with deletion of sequence coding for the entire glycine rich region or the amphipathic alpha-helical region into a het-cPA strain gave compatible transformants, suggesting that both the glycine rich region and the amphipathic alpha-helical region are required for mediating vegetative incompatibility (Saupe et al., 1996b). Introduction o f a het-cOR allele with deletion of the sequence coding for the signal peptide into a het-cPA strain gave incompatible transformants (S. J. Saupe and R. Todd, personal communication). The disruption of het-cOR does not affect the vegetative growth or fertility o f TV. crassa, but strains containing an inactivated het-cOR allele display dual compatibility with both het-cOR and het-cPA strains (Saupe etal., 1996b) The three het-c alleles encode similar peptides, which share approximately 85% amino acid sequence identity. Sequence comparison revealed three variable regions between the three H E T - c polypeptides. The first variable region, from amino acid positions 149-212 in H E T - c 0 R , has 78% amino acid sequence identity between H E T - c 0 R , H E T - c P A and H E T - c G R * OR The second variable region (region n, Figure 1-7), from positions 247-284 in H E T - c , showed only 45% sequence identity between the three polypeptides. Within this variable region, the H E T - c polypeptides differed by 20 amino acid substitutions and three deletion or insertion events (Figure 1-8). The third variable region, from positions 487-610 in H E T - c 0 R , shares 96% sequence identity between H E T - c 0 R and H E T - c P A , while the identity between H E T - c 0 R or H E T - c P A with H E T - c G R was only 68%. Chimeric construction and transformation assays demonstrated that the variable region U (position 247 to 284 in H E T - c 0 R ) controls het-c allelic specificity (Saupe and Glass, 1997) 31 s i S n a l specificity peptide region coiled-coil glycine-rich domain HET-c O R 1-31 149-212 247-284 426^58 487-610 708 966 aa HET-c O R I II I I I HET-c P A HET-c G R Figure 1-7. Features of deduced H E T - c peptides from the D N A sequences. The H E T - c is a putative peptide o f 966 amino acids, including a signal peptide at the N-terminal, an amphipathic alpha helical (coiled-coil) motif and a glycine rich domain at the C-terminal (Saupe et al., 1996b). The deduced three peptides H E T - c O R , H E T - c P A and H E T - c G R share more than 80% amino acid identity and differ in three variable domains, I, II and III (Saupe and Glass, 1997). Chimeric construction suggested that the variable domain II (amino acid 247-284 in H E T - c 0 R ) confers het-c allelic specificity (Saupe and Glass, 1997). EDFPAHSNYCELVLIDMEERRGGH-SPVFPHVGTDTRITLRNDTRNNG KSVWPLVTGT OR EDFPAHSNYCELALIDIHEKETRSESRIFPHVGTATRITL NNG KLVWPLVTGT GR EDFPAHSNYCELVLIDMEERRGGH-SPVFPHVGTATKLKL ENRQFRRVRPGEGYDSGAKYAWPLVTGT PA * * * * * * * * * * * * *** * ****** * * * * ******* Figure 1-8. Amino acid sequence comparison o f the variable region II in HET-c . Note that the three polypeptides differ by several amino acids and the pattern of insertions/deletions. H E T - c 0 R has a putative insertion of five amino acids in length; H E T - c P A has a putative insertion of 15 amino acids in length. Asterisks indicate identical sites. 32 1.5 THESIS OBJECTIVES Vegetative incompatibility is a universal phenomenon among filamentous fungi. However, the biological significance of vegetative incompatibility in fungal populations is unclear. It has been proposed that vegetative incompatibility may be a fungal "immune" system. The question whether vegetative incompatibility is a bona fide non-self recognition system and whether it confers a selective advantage for non-self recognition, such as the MHC complex in vertebrates and the S system in plants, is unclear. In N. crassa, the het-c locus has been molecularly well characterized, and polymorphisms at the het-c locus have been observed in a number of N. crassa isolates (Saupe and Glass, 1 9 9 7 ) . The link between het-c polymorphisms and allelic specificity allowed me to examine the evolutionary features of the het-c locus and thus shed light on the biological significance of vegetative incompatibility. Morphological aspects associated with vegetative incompatibility have been extensively documented in 7Y. crassa (Perkins, 1 9 8 8 ; Jacobson et al., 1 9 9 8 ) . However, the molecular mechanisms behind vegetative incompatibility remain largely unknown. The isolation of naturally occurring het-c alleles in the study of het-c evolutionary features provided tools for the dissection of the molecular basis of allelic specificity. However, an understanding o f how amino acid and spatial variability in the specificity domain affects vegetative incompatibility does not address the question of how non-self recognition is mediated. The molecular basis of non-self recognition is well understood in the yeast mating system. Is non-self recognition during vegetative incompatibility mediated via H E T - c heteromeric complex formation as the case for a l / a 2 in S. cerevisiae? 33 In the effort to understand the biological significance and the molecular mechanisms of vegetative incompatibility, the specific objectives of my thesis were: 1. A n examination o f the evolutionary features of the het-c locus in the Sordariaceae to define the biological significance of vegetative incompatibility in fungal populations. 2. To delineate the determinants for het-c allelic specificity. 3. To determine whether non-self recognition is mediated via the mechanism of H E T - c heteromeric complex formation. In Chapter 2,1 analysed the evolutionary feature of the het-c locus in the Sordariceae. The evolutionary features between the het-c locus and known loci responsible for non-self recognition, such as the MHC and the S loci , were compared. In Chapter 3, specificity of the naturally occurring het-c alleles that were isolated in Chapter 2 was assayed to examine whether amino acid substitutions in the variable domain of H E T - c primarily affect het-c specificity. Since H E T - c O R , H E T - c P A and H E T - c G R in N. crassa also differ by deletions/insertions, artificial het-c alleles were generated to examine how the deletions/insertions may affect het-c specificity. A model of how het-c specificity may be determined is proposed. In Chapter 4,1 performed immunoprecipitation assay to determine whether the H E T - c proteins form heteromeric complexes during vegetative incompatibility. A possible model of het-c non-self recognition is proposed. 34 Chapter 2 Evolutionary analyses of the het-c locus "This polarity between the Self-Assertive and Integrative tendencies is inherent in the concept of hierarchic order; and a universal characteristic of life." - Arthur Koestler, The Ghost in the Machine. Some sequences in this chapter were contributed by Dr. Sven Saupe (presently at Universite de Bordeaux, Bordeau, France) and also published in Saupe and Glass, 1997. Most o f the results described here are published in W u J., S. J. Saupe and N . L . Glass (1998). 2.1 INTRODUCTION The phenomenon of vegetative incompatibility is widespread in filamentous fungi, however, its biological significance in natural populations is unclear. Todd and Rayner (1980) suggested that vegetative incompatibility is a protection mechanism of the "fungal individual" against invasion of foreign nuclei and cytoplasm. In a population genetic study, Nauta and Hoekstra (1994) suggested that selection against transmission of a harmful cytoplasmic element is a more plausible explanation for vegetative incompatibility than against a nuclear gene invasion. Vegetative incompatibility has been shown to restrict horizontal transmission of cytoplasmic elements between individuals in several species, such as TV. crassa (Caten, 1972; Debets et al., 1994), Cryphonectriaparasitica (Anagnostakis, 1983) and Aspergillus niger (Van Diepeningen et al., 1997). It has been argued whether vegetative incompatibility in fungi functions as a non-self recognition system or whether its existence is a simple consequence of variation in genes with critical cellular functions (Begueret et al., 1994). I f vegetative incompatibility confers non-self recognition function in fungal populations, selection may favor polymorphisms at het loci . Therefore, het loci should exhibit hallmarks o f loci under balancing selection and 35 display similarities to the MHC and S loci , such as large number o f alleles existing in a population, persistence of trans-specific polymorphisms and enhanced nonsynonymous nucleotide substitutions at polymorphic sites. In N. crassa, het-c encodes a putative glycine rich polypeptide containing a consensus sequence for a signal peptide. A 34-48 amino acid region of H E T - c determines het-c allelic specificity (Saupe and Glass, 1997). The identification o f a particular polymorphic domain that confers het-c allelic specificity has allowed us to examine the diversity of het-c allelic types at the het-c locus in different species within the Sordariaceae and to derive inferences about the evolutionary history of selection in the het-c gene. 2.1.1 Trans-specific polymorphism Trans-specific polymorphism refers to a peculiar pattern in which allelic lineages (or gene polymorphisms) have been passed from an ancestral species to its descendent species (Klein et al., 1993). In phylogenetic analyses, trans-specific polymorphisms are inferred when an allele from one species is found to be more closely related to an allele from another species or genus than to other alleles from the same species; that is, alleles cluster in phylogenetic analyses rather than species. Observations o f trans-specific polymorphisms have often been restricted to loci believed to be under balancing selection. Two such systems have been studied extensively, the major histocompatibility complex (MHC) o f vertebrates (for reviews, see Kle in , 1986; K l e i n et al., 1998; Hughes and Yeager, 1998) and the self-incompatibility S loci in flowering plants (for reviews, see K l e i n et al., 1998; Charlesworth and Awadalla, 1998). However, neutral trans-specific polymorphisms may occur under other circumstances, such as a relatively short period since speciation and a large founding 36 population size. Such examples have been documented in the sequences of 3 ' U T R or introns of five randomly selected nuclear genes in the Lake Victoria haplochromines (Nagl, 1998). The mammalian MHC loci are good examples of trans-specific polymorphism (Ayala, et al., 1994). A s an example, in phylogenetic analysis o f the MHC class II DRB alleles, the human allele HLA-DRB1 *0302 clustered with the chimpanzee allele Patr-DRB1 *0305. The alternative human allele HLA-DRB1 *0701 clustered with the chimpanzee allele Patra-DRB *0702. The human allele HLA-DRB1 *0302 differs from the chimpanzee allele Patr-DRBl *0305 in the exon encoding the peptide binding region (PBR) by 13 nucleotide substitutions and from the other human allele HLA-DRB1 *0701 by 31 nucleotide substitutions, while the chimpanzee allele Patra-DRB*0702 differs from the human allele DRB*0701 by only two nucleotide substitutions. This relationship is reflected in a phylogenetic tree o f the four DRB alleles (Figure 2-1). Phylogenetic analysis of DRB alleles indicated that the DRB allelic lineages arose prior to the separation o f the human and chimpanzee lineages and have persisted in subsequent populations since their divergence. Trans-specific polymorphism at the MHC loci has also been described in other mammals such as Old Wor ld primates and apes (Boyson et al., 1995,1996; Cadavid and Watkins, 1997; for review, see Cadavid et al., 1997) and other vertebrates including birds (Bourlet et al., 1988), amphibians (Flajnik et al., 1991) and fish (Hashimoto et al., 1990; Graser et a l , 1996). The trans-specific nature of the S locus polymorphism has been demonstrated for both the gametophytic (Ioerger et al., 1990; Richman et al., 1996) and the sporophytic (Dwyer et al., 1991) self-incompatibility systems. In a sample of S alleles from the gametophytic self-incompatibility system in the Solanaceae (Figure 2-1, Ioerger et al., 1990), some S alleles of 37 Nicotiana alata clustered with certain S alleles of Petunia inflata rather than with other S alleles from other N. alata isolates. For example, the Si allele o f P. inflata clustered with SFu allele of N. alata; and the S3 allele of P. inflata clustered with the Sz allele o f N. alata. The S allele clustering pattern suggests that S allele polymorphisms arose prior to the divergence o f P. inflata and N. alata. MHC Human HLA 0302 Chimpanzee Patra 0305 Human HLA 0701 Chimpanzee Patra 0702 Slocus SF11 Nicotiana alata SI Petunia inflata Sz Nicotiana alata S3 Petunia inflata Figure 2-1. Examples of trans-specific polymorphism at the MHC DRB1 locus and plant self-incompatibility S locus. The allelic lineages o f four alleles at the MHC DRB 1 locus from humans (HLA) and chimpanzees (Pan troglodgtes, Patr) predate the divergence of the human and chimpanzee lineages (Adapted from Aya la et al., 1994). Similarly, the allelic lineages of four S alleles predate the divergence o f Nicotiana alata and Petunia inflata (modified from Ioerger et al., 1990). Note that many more alleles are present within the populations than are shown here. 3 8 2.1.2 Selection systems Mutations can be selectively neutral, deleterious, or advantageous (reviewed in Kle in et al., 1998). A n allele with negative selective value (disadvantage) is usually eliminated from the gene pool soon after its appearance. A n allele with an initially positive selective value can also be lost, but i f it isn't, it can either become fixed (in the case of directional selection) or established at an immediate equilibrium frequency (in the case of balancing selection, see below). A neutral allele w i l l eventually become eliminated or fixed (i.e., replace all other alleles at that locus) by genetic drift. Most neutral polymorphisms are not expected to persist in populations for a very long time. Theoretical studies predict that a neutral allele, i f not lost, takes an average o f 4Ne generations to become fixed, where Ne is the effective population size (which is roughly the number of gene pairs at a given locus that are passed on from one generation to the next) (Kimura, 1983; Ne i , 1987). Assuming a long-term effective population size of 10 4 and generation time of 15 years for humans, it w i l l take only 600, 000 years for a neutral allele to become fixed. Thus, neutral polymorphism is a relatively transient phenomenon with respect to evolutionary time. If the allele occurs in the vicinity of a site under balancing selection, neutral alleles may persist in a population for much longer periods o f time than expected based on the attributes of the population (i. e., a fast speciation or a oversized population) (Nagl, 1998). Genetic theory predicts that balancing selection slows down or completely hinders the fixation o f alleles and thus allows the persistence of allelic polymorphisms over extensive periods o f time (Maruyama and Ne i , 1981). Under balancing selection, allele polymorphism can persist without fixation for as long as selection pressure persists and the population 39 remains at a reasonable size (Takahata and Ne i , 1990). Two forms of balancing selection have received a great deal o f attention in the literature, namely, overdominant selection and frequency-dependent selection. In the case of overdominant selection, heterozygotes have a selective advantage over either homozygote. In the case o f frequency-dependent selection, an allele is advantageous when it is rare but becomes disadvantageous when common. Balancing selection is the most efficient mechanism to maintain polymorphisms and trans-specific allele lineages (Takahata, 1990). A high level o f polymorphism in both the MHC and S loci is believed to be maintained by balancing selection (Doherty and Zinkernagel, 1975; Hughes and Ne i , 1988; 1989). A t the MHC class I DRB locus, trans-specific allele lineages have persisted for at least 29 mil l ion years in humans, 36 mil l ion years in chimpanzee, 34 mil l ion years in gorillas and 46 mil l ion years in macaques (Klein, 1986; Mayer et al., 1988; McConnel l et al., 1988; Lawlor et al., 1988; Aya la et al., 1994). A t the gametophytic self-incompatibility S loci , trans-specific allele lineages have been retained for at least 36 mil l ion years (Ioerger et al., 1990). It is unclear whether selection at MHC loci is overdominant (Doherty and Zinkernagel, 1975; Maruyama and N e i , 1981) or frequency-dependent (Takahata and Ne i , 1990), or some Other form o f selection (e. g., maternal-fetal interactions) (Clarke and Kirby, 1966; Ohta, 1991; Hughes and Ne i , 1992a). Polymorphism at the S locus is believed to be maintained primarily by frequency-dependent selection (Vekemans and Slatkin, 1994; Schierup et al., 1997). 4 0 2.1.3 Synonymous and nonsynonymous nucleotide substitutions There are two classes of nucleotide substitutions in protein coding regions: synonymous and nonsynonymous. Synonymous (or silent) substitutions are nucleotide substitutions that do not result in amino acid changes, whereas nonsynonymous nucleotide substitutions are those that change amino acids within a protein. The synonymous nucleotide substitution rate largely reflects the mutation rate. Theoretical studies predict that overdominat selection should enhance nonsynonymous nucleotide substitutions (Lopez et al., 1982). A n excess of nonsynonymous nucleotide substitutions was observed in both the P B R coding region o f MHC molecules (Nei and Gojobori, 1986; Cadavid et al., 1997; Hughes and Ne i , 1989) and the polymorphic sites o f the S alleles (Hinata et al., 1995; Clark and Kao, 1991). 2.1.4 Hypotheses on the biology of vegetative incompatibility Natural populations of N. crassa are highly polymorphic for het genes (Mylyk, 1975; Debets et al., 1994; 1998). Two hypotheses have been proposed for the biological significance of vegetative incompatibility in relationship to the polymorphisms of het genes in fungal populations. One proposes that vegetative incompatibility is a bona fide non-self recognition system in fungi. Under this hypothesis, incompatibility systems have been selected to prevent heterokaryon formation between genetically unlike individuals (non-self) to limit the horizontal transfer of infectious elements, such as mycoviruses, debilitated organelles and deleterious plasmids (Caten, 1972; Lane, 1981). The alternative hypothesis is that vegetative incompatibility is a consequence of genetic variation in genes with critical 41 cellular functions in which heteroallelism (co-existence o f two allelic types) is unfavorable and that vegetative incompatibility is not directly favored (reviewed in Begueret et al., 1994). The two hypotheses imply different evolutionary features of het genes as depicted in Figure 2-2. Under the first hypothesis, selection favors polymorphisms at het loci for non-self recognition to prevent the invasion o f unfavorable cytoplasmic elements from unlike individuals. In this case, alleles at het loci should exhibit evolutionary features similar to other loci that regulate non-self recognition, such as MHC and S loci , i.e. long persistence of polymorphisms in allelic lineages. Under the second hypothesis, polymorphisms at het loci could be found transiently within populations due to random mutations but are not maintained by selection over long periods of time. Thus, one would expect monophyletic relationships of het genes within contemporary species. To distinguish between these two hypotheses, I describe the evolutionary features of the het-c locus in species in the Sordariaceae. Evidence that supports balancing selection acting at the het-c locus includes the persistence o f trans-specific polymorphisms and excess nonsynonymous nucleotide substitutions at the polymorphic region in the het-c specificity domain. The study supports the hypothesis that vegetative incompatibility is under balancing selection, which is consistent with its role as a non-self recognition system. 42 monophyly trans-specific polymorphism Figure 2-2. Schematic diagram illustrating the effect o f balancing selection when alleles are descended from an ancestral allele pool. Patterns o f dots do not indicate different alleles, but rather different modern species found carrying alleles. One would expect that alleles within a species are more closely related i f alleles are neutral (left); and that functionally related alleles in different species should be more closely related than alleles within a species (trans-specific polymorphism) i f balancing selection has been acting (right). 43 2.2 MATERIALS AND METHODS 2.2.1 Strains and media For the het-c phylogenetic analysis, we used 40 strains representing eleven species and three different genera within the Sordariaceae (Table 2-1). A l l strains were obtained from Fungal Genetics Stock Center ( F G S C ; Department of Microbiology, University of Kanas Medical Center, Kansas City, K S ) . A l l strains were maintained on Vogel ' s vegetative growth media (Vogel, 1964). 2.2.2 Genomic DNA isolation and Southern analysis Genomic D N A was prepared as described by Oakley et al. (1987). Two micrograms of genomic D N A was digested with restriction enzymes Pst I and Sac I according to the manufacturer's instructions. Ge l electrophoresis and transfer o f D N A to nylon filters (Nytran + , Schleicher & Schuell, Keene, N H ) was done according to Sambrook et al. (1989). A 3.9 kb fragment o f het-c0R genomic clone was radiolabeled with 3 2 P using the T7 quick primer kit (Pharmacia, Baie d'Urfe, PO). 44 Table 2-1. Strains used for phylogenetic analysis. Strain designation FGSC number Origin Source of sequences het-c type Neurospora crassa 4711 Haiti Saupe and Glass, 1997 PA N. crassa 4832 Ivory Coast S. J. Saupe PA N. crassa 2489 Louisiana S. J. Saupe OR N. crassa 1455 Unknown Saupe and Glass, 1997 GR N. crassa 4709 Haiti S. J. Saupe OR N. crassa 430 Ivory Coast Saupe and Glass, 1997 PA N. crassa 847 Louisiana Saupe and Glass, 1997 OR N. crassa 1693 Louisiana Saupe and Glass, 1997 OR N. crassa 1824 Pakistan Saupe and Glass, 1997 OR N. crassa 4481 Louisiana This study OR N. crassa 4486 Louisiana This study PA N. crassa 4499 Louisiana This study GR N. crassa 1945 Florida Saupe and Glass, 1997 GR N. crassa 967 Liberia Saupe and Glass, 1997 GR N. intermedia 2316 Florida S. J. Saupe PA N. intermedia 1940 Florida S. J. Saupe OR N. intermedia 3290 Oahu S. J. Saupe PA N. intermedia 3721 Kauai S. J. Saupe OR N. intermedia 1799 Malaya S. J. Saupe OR N. discreta 3228 Texas S. J. Saupe PA N. discreta 5923 Texas S. J. Saupe X N. discreta 6788 New Guinea This study GR N. discreta 6793 Brazil This study OR N. sitophila 963 France This study OR N. sitophila 5940 Tahiti S. J. Saupe OR N. sitophila 5941 Tahiti S. J. Saupe PA N. tetrasperma 6538 Tahiti S. J. Saupe PA N. tetrasperma 1270 Unknown S. J. Saupe PA N. pannonica 7221 Hungary S. J. Saupe PA N. dodgi 1692 Puerto Rico S. J. Saupe GR Gelasinospora sp. 8241 Yucatan Penn. This study OR Gelasinospora sp. 8239 Yucatan Penn. This study OR Gelasinospora sp. 8243 Yucatan Penn. This study PA Gelasinospora sp. 8242 Yucatan Penn. This study OR Sordaria brevicollis 7140 New York Zoo This study GR S. brevicollis 1903 New York Zoo This study OR S. heterothallis 2738 Texas This study PA S. heterothallis 2739 Texas This study OR S. sclerogenia 2741 Ceylon This study GR S. sclerogenia 2740 Ceylon This study PA 45 FGSC, Fungal Genetics Stock Center. N. crassa, Neurospora crassa; N. discreta, Neurospora discreta; N. sitophila, Neurospora sitophila; N. intermedia, Neurospora intermedia; N. tetrasperma, Neurospora tetrasperma; N. pannonica, Neurospora pannonica; N. dodgei, Neurospora dodgei; S. brevicollis, Sordaria brevicollis; S. heterothallis, Sordaria heterothallis; S. sclerogenia, Sordaria sclerogenia. P A , O R and G R refer to het-c , het-c and het-cGR allelic specificity type as designated in Neurospora crassa (Saupe and Glass, 1997). X, unidentified specificity. 46 2.2.3 Polymerase Chain Reaction (PCR) amplification of het-c specificity region Genomic D N A was used for P C R amplification. The region of het-c allelic specificity was amplified with primers designed from the sequence o f the het-cOR allele (Saupe et al., 1996b). Positions o f the primers in the het-c sequences are shown in Figure 2-3. The primers are: Red, 5 ' ( 5 9 8 ) G G A G A C A T G G C G A T A T C G ( 6 1 5 ) 3 ' ; Ye l low, 5 ' ( 1 4 4 1 ) G T G A G G C A C A A C C C A C T C ( 1 4 2 4 ) 3 ' . P C R reactions were prepared in 25 p i volumes including 100 ng of genomic D N A , 10 m M T r i s - H C l (pH 8.3), 50 m M KC1, 1.5 m M M g C l 2 , 0.2 m M each of d A T P , dTTP, dCTP and dGTP, 5 pmol of each primer, and 2.5 unit of Taq D N A polymerase (Boehringer Mannheim, Indianapolis, IN). Amplification cycles were performed under the following conditions: 95°C for 5 min followed by 30 step cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and 72°C for 10 min after the last cycle. 2.2.4 Subcloning of PCR products P C R products were subcloned into the pCRII vector using the T A cloning kit (InVitrogen, San Diego, C A ) . Ligation reactions were performed according to the manufacturer's guide. Subcloning efficiency competent Escherichia coli DH5a cell (F, endAl, hsdR17, supE44, lacZmlS) was used for transformation. Plasmids were prepared using Qiagen miniprep kits (Qiagen, Chatsworth, California). 47 48 The D N A sequence of P C R products was determined using the A B I Taq DyeDeoxi Terminator cycle method (Mississauga, ON) on an A B I 373 automatic sequencer at the N A P S (Nucleic A c i d and Protein Service) unit, Biotechnology Laboratory, University of British Columbia. Approximately 500 ng o f purified plasmid containing the expected P C R product was used as a template in a total of 20 ui reaction mixture containing 3.2 pmol primer (Red or Yel low; see section 2.2.3). The sequencing reaction was performed in a Perkin-Elmer thermal cycler for a total of 25 cycles as follows: 96°C for 30 sec, 50°C for 15 Sec, 60°C for 4 min. 2.2.6 Sequence analysis The D N A sequences were translated into amino acid sequences using universal genetic codons with the program Mac Vector 4.1 (International Biotechnologies, Inc., Tecnomara, Fernwald, Germany), aligned using ClustalW 6.1 (Institute for Biomedical Computing, Washington University, St. Louis, M O ) , and adjusted manually according to positional homology. D N A sequences of the het-c specificity region from the isolates listed in Table 2-1 were also aligned by ClustalW 6.1 and modified manually according to the corresponding amino acid sequence positional homology. Insertions or deletions were positioned according to the amino acid alignments. 2.2.7 Phylogenetic tree reconstruction The phylogenetic tree of het-c was constructed using P H Y L L P 3.0 (Felsentein, 1993). Three different methods, the maximum parsimony method (Swofford, 1990), the neighbor-49 joining method (Saitou and Ne i , 1987) and the maximum likelihood method (Felsenstein, 1981), were used for tree reconstruction. Parsimony methods search for the tree with the fewest evolutionary changes (e. g., the smallest number of nucleotide changes in the case o f D N A sequence data) or steps (transformations from one character state to another) and are not based on explicit evolutionary models (i.e, there are not specific parameters or assumptions for nucleotide substitution probabilities) for computation. Since no explicit evolutionary model is assumed with parsimony, the phylogenetic trees can be inconsistent (Felsenstein, 1988). However, the computation time is short with parsimony methods. The neighbor-joining method is based on distance calculation between species (Saitou and Ne i , 1987). For nucleotide sequence data, the distances are calculated from the fraction of sites differing between two sequences and using specified nucleotide "transversion vs. transition ratio" corrections. The algorithm makes a prediction o f the distance for each pair as the sum of branch lengths in the path from one species to another through the tree. Neighbor-joining does not check the possibility of alternative trees, thus it is difficult to see how different kinds of discrepancies from a tree are weighted. The algorithm is very fast, it is particularly effective for large studies or for bootstrap resampling studies. Maximum likelihood methods produce consistent results i f the model is correct. However, with many taxa, it is difficult to search exhaustively for the maximum likelihood tree. Max imum likelihood can estimate the parameters (e. g. transversion/transition rate, variation o f substitution rate from site to site) of the model from the data. Maximum likelihood provides a way of handling missing or ambiguous data, as the probability of observing each of the bases allowed by the ambiguity can be explicitly evaluated. Maximum 50 likelihood considers that nucleotide changes are more l ikely along long branches than short ones and estimation o f branch lengths is an important component. The method of computing maximum likelihood can be very slow, and large amounts of computation time are required for trees with many species or bootstrapping. In parsimony tree reconstruction, gap sites were treated as a fifth character, with a deletion or insertion motif treated as a single event. In neighbor-joining tree reconstruction, distances were estimated using the Dnadist program in P H Y L L P 3.0 with Kimura's two-parameter correction (Kimura, 1980), and gap sites were eliminated in all pairwise distance calculations. In maximum likelihood tree reconstruction, regions of deletion were treated as "missing data". The robustness of the dendrogram topology was determined by bootstrap with 500 replicates for both parsimony and neighbor-joining trees (Felsenstein, 1985). 2.2.8 Calculation of synonymous and nonsynonymous nucleotide substitutions Number of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (ds) was calculated by pairwise comparison of sequences from all isolates with the program M E G A (Kumar et al., 1993) according to Juke-Cantor's correction. Gap sites were treated according to the "pairwise-deletion" option in the M E G A program, which ignores the gaps present in pairwise comparisons of sequences. Microsoft Excel was used to map the substitutions to each codon position. 51 2.2.9 Statistical analyses A standard z-score test (Capon, 1988) was used to test the following hypotheses at the P<0.05 significant level: 1) mean d s = mean d N ; 2) mean d N in het-c polymorphic region (the region of the 34 - 48 amino acids which confers het-c allelic specificity) = mean d N in het-c non-polymorphic region; 3) the ratio of d N : d s in the het-c polymorphic region = the ratio of d N : d s in the het-c non-polymorphic region. Values of d N : d s ratios and the associated standard errors were calculated using the delta method (Kotz and Johnson, 1989; Haydon et al., 1998). 52 2.3 RESULTS 2.3.1 Genomic Southern blot analysis To examine i f the D N A sequence of het-c is conserved in the Sodariaceae, genomic D N A from 15 isolates, including TV. crassa, N. discreta, N. sitophila, N intermedia, S. sclerogenia and S. brevicollis, was subjected to Southern blot analysis probed with a 3.9 kb fragment of the het-c0R genomic clone. Under conditions of both low and high stringency, only a single band corresponding to het-c was detected in the genome o f all the isolates analysed (Figure 2-4). This result indicates that het-c is highly conserved in different Neurospora and even Sordaria species, and that het-c is present in a single copy in these isolates, as was previously determined for TV. crassa strains (Saupe et al., 1996). 2.3.2 PCR products and DNA sequences To examine evolutionary features of het-c, genomic D N A from all the isolates was amplified by P C R using primers spanning the het-c specificity region of TV. crassa (Figure 2-3) and the P C R products were sequenced. A single band was produced in each P C R reaction with the expected size of 830 bp to 860 bp (data not shown). The D N A sequences of the het-c specificity region differed among these isolates (see Appendix 7.1), even within a single species. The two most distantly related alleles of all the isolates, Np7221 and Sh2739, showed only 66% nucleotide identity. 53 Figure 2-4. Southern hybridization of genomic D N A from different species and genera (listed in Table 2 .1) to a plasmid carrying the het-c0R clone from N. crassa. Genomic D N A was digested with Sac I and Pst I. Sequences corresponding to het-c are apparently present as a single copy in all tested isolates. 54 2.3.3 Amino acid sequence of the het-c specificity region Although the D N A sequences of the het-c specificity region varied among the isolates, the inferred amino acid sequences from these isolates fell into one of the three H E T -c specificity types based on the pattern of insertion/deletions as first observed in the N. crassa het-c alleles (Figure 2-5, Saupe and Glass, 1997). A comparison of the H E T - C peptide sequences from all the isolates showed conserved and variable domains. Regions flanking the variable domain showed a high level of amino acid identity among all the isolates. Two amino acid blocks, I and II, are highly polymorphic among het-c alleles (Figure 2-5). The polymorphic block I contains the sequences of M E E R R G G ( Q ) H and ffl(Q)EKET(D)R(G/C)S that are found in both H E T - c P A and H E T - c O R groups. For all species containing a H E T - c G R - type allele, the polymorphic block I contains only a similar sequence of fflEK(N)ET(N)R(C,P)S. Partial duplication analysis of the two N. crassa strains, Ncl824 and Nc2489, indicated that amino acid sequence composition in the polymorphic block I does not affect het-c specificity (Saupe and Glass, 1997). The second polymorphic block (II) contains the sequences that are divergent among all het-c allelic types. H E T - c 0 R - type alleles contains a sequence motif of T A ( V , D ) T R V ( I ) T L in block II, while H E T - c G R - type alleles contain a sequence motif of TA(V)TR(Q) ITL , with the exception of Ndo1692, which contains the sequence G A H T R K T L . The polymorphic block II in H E T - c P A - type of alleles contain sequences of T A ( E , R ) T K L K L ( V , R ) or A H T R L R ( K , T ) Y ( L ) . Chimeric studies (See chapter 3) demonstrated that the sequence diversity in the polymorphic block II also does not affect het-c specificity. A five amino acid block, F P H V G , located between the two polymorphic blocks is conserved among all the isolates. 55 Across species and alleles, H E T - c peptide sequence variation in the specificity motif (Figure 2-5) occurred at 43 out of 91 amino acid residues. With in H E T - c 0 R - type alleles, the variation occurred at fourteen out of 74 amino acid residues. With in H E T - c G R - type alleles, the variation occurred at thirteen out of 70 amino acid residues. With in H E T - c P A - type alleles, the variation occurred at 41 out of 85 amino acid residues. The fifteen to sixteen amino acid sequences within the het-cPA specific insertion region IR2 (Figure 2-5) are the most polymorphic. A n exceptional allele, Ndi5923, was found in N. discreta, which has a deletion o f five amino acid residues in the het-cPA - type specific insertion motif. Wi th the exception of the pseudohomothallic species, N. tetrasperma, at least two of the three het-c allelic variants were found in all species with more than one isolate. A s shown in Figure 2-5, in the heterothallic species, S. brevicollis, the two inter-fertile isolates from the New York Zoo each contained a het-c allele of a different H E T - c type, H E T - c 0 R (Sbl903) and H E T - c G R (Sb7140). The two S. sclerogenia inter-fertile isolates from Ceylon also have different H E T - c allelic types, one showing H E T - c P A type (Ss2740) and the other showing H E T - c G R - type (Ss2741). Two different H E T - c allelic types, H E T - c 0 R (Sh2739) and H E T -c P A (Sh2738) were also found in the two inter-fertile S. heterothallis isolates. Similarly, two alternative het-c allelic types, H E T - c P A and H E T - c O R , were found in the four inter-fertile Gelasinospora sp. isolates (Gsp 8243, Gsp8241, Gsp8239 and Gsp8242). A t least two or all three of the het-c allelic types were identified in the heterothallic Neurospora species, N. crassa (Nci 130, Nc4711, Nc4832, Nc4486, Ncl945, Ncl455, Nc967, Nc4499, Nc4481, Ncl824, Nc2489, Nc847, Nc4709, Ncl693), N. sitophila (Ns5941, Ns 963, Ns5940), N intermedia (N12316, N13290, M3721, Nil940, Nil 799), and TV. discreta (Ndi3228, Ndi5923, Ndi3268, Ndi6788, Ndi6793). 56 In summary, an amino acid sequence comparison of the het-c specificity region from all o f the 40 isolates showed that two to five different amino acid residues were found at each polymorphic site from different isolates in the H E T - c specificity domain, in particular within the two polymorphic blocks and the H E T - c P A - specific insertion motif. 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J J i J < < < a o a o o o J J j CJ CJ u Oi P i Oi J i J J < < < MM Pi Pi Pi fa fa fa P P P WWW •J pj J IG IG IG •5 i J J A < < a a a o o o J J J CJ cj CJ Di Oi Di J J J < < < H H H Pi PI fa fa Q P W H J J XX J J «< «: O I o 0 o J j CJ u 01 Oi i J J << << H H H H CJ O s s ca ca 9 9 P. P . fa fa p a H w J j S IG J j < «: o a o o ]-58 Figure 2-5. Inferred amino acid sequences of P C R amplified het-c allelic specificity motif from 40 isolates. The alignment was obtained by ClustalW 6.1 and modified manually. Nc, Neurospora crassa; Ndi, Neurospora discreta; Ns, Neurospora sitophila; Nt, Neurospora tetrasperma; Np, Neurosporapannonica; Ndo, Neurospora dodgei; Ss, Sordaria sclerogenia Sh, Sordaria heterothallis; Sb, Sordaria brevicollis; Gsp, Gelasinospora sp. Numbers following indicate the Fungal Genetics Stock Center (FGSC) number or P number (4481, 4486, 4499 strains). P A (Panama), G R (Groveland), and O R (Oakridge) refer to the three molecularly characterized alleles that confer different het-c specificity (Saupe and Glass, 1997). Asterisks and dashes indicate identical and deletion residue sites, respectively. Underlined section is het-c specificity variable region. Polymorphic block I and II, putative OR-specific insertion sequence (IS1) and PA-specific insertion motif (IS2) are indicated. 5 9 2.3.4 Trans-specific polymorphism of het-c Sequence analysis of the P C R products of the het-c specificity region from 40 natural isolates showed 39 distinct alleles based on nucleotide sequences (Nci 130 and Nc4711 are identical; Appendix 7.1); 24 alleles had distinct amino acid sequences (Figure 2-5). In order to examine the relationship of the het-c alleles, I performed a phylogenetic analysis of D N A sequences o f the het-c specificity region using the neighbor-joining algorithm based on Kimura's two-parameter distances (Figure 2-6, Figure 2-7), parsimony (Figure 2-8, Figure 2-9), and maximum-likelihood (Figure 2-10, Figure 2-11). Phylogenetic trees were reconstructed under two conditions: including and excluding the allelic specific insertion/deletion sequences (sequences coding for IS1 and IS2 in Figure 2-5). Thirty-one sequences isolated from seven Neurospora species (N. crassa, N. discreta, N. intermedia, N. sitophila, N. tetrasperma, N. pannonica and N. dodgei), five sequences from three Sordaria species (S. brevicollis, S. heterothallis and S. sclerogenia) and three sequences from Gelasinospora sp. were analysed. Phylogenetic analysis showed that het-c subgrouped by allelic type rather than according to species - a trans-specific mode of polymorphism. In all the trees reconstructed using D N A sequences that include the insertion/deletion sites in the het-c specificity region, the clustering pattern of het-c alleles was supported by robust bootstrap values. Five hundred bootstrap replicates were analysed using both neighbor-joining (Figure 2-6) and parsimony (Figure 2-8) methods. Eighty-five percent of neighbor-joining trees and 100% of parsimony trees supported the clustering of the eleven het-cOR - type alleles, Nc2489, Nci 693, Nc4709, Nc847, Nc4481, Gsp8242, Sh2739, Nil940, Nil 799, Ns963 mdNs5940. A l l the trees (100%) constructed using neighbor-joining and parsimony methods supported the clustering of the 60 four het-cUK - type alleles, Sbl903, Ndi6793, Gsp8239 and Gsp8241. Ninety - three percent of neighbor-joining trees and 99% of parsimony trees supported the subgrouping of the nine het-cPA - type alleles, Ndi3228, Nc4486, Nc4711, Nci 130, NU270, Nt6583, Ni2316, N13290 and Nc4832. Ninety-five percent of neighbor-joining trees and 92% of parsimony trees supported the clustering of the three het-cPA - type alleles, Ss2740, Np7221 and Gsp8243. Eighty-percent o f the neighbor-joining trees and 87% of the parsimony trees supported the subgrouping of seven het-cGR - type alleles, Nc967, Nc4499, Nci455, Ss2740, Sb7140, Ndi6788 and Nci945. A similar grouping of het-c alleles was also observed in the tree reconstructed using maximum likelihood (Figure 2-10). In each of the groups, alleles were found from at least two species. It is possible that the clustering of het-c alleles is affected by the D N A sequence alignment bias based on the placement of the insertion/deletion sites. To examine this possibility, trees were reconstructed using D N A sequences excluding the deletion/insertion sites. Although certain differences in the subgroup branching points and weaker bootstrap support were observed, the subgroupings of het-c alleles were consistent in all trees. In 500 neighbor-joining trees (Figure 2-7), 99% of the replicates supported the subgrouping of the fifteen het-cOR - type alleles from seven species and three genera and the subgrouping of the ten het-cPA - type alleles from five Neurospora species; 92% of the replicates supported the subgrouping of the five het-c - type alleles from three species; and 94% of the replicates supported the subgrouping of the three het-cPA - type alleles, Gsp8243, Ss2740 and Np7221. In 500 parsimony trees (Figure 2-9), 85% of the replicates supported the subgrouping of the three het-cPA - type alleles, Gsp8243, Ss2740 and Np7221; 98% of the replicates supported the subgrouping o f the four het-cOR - type alleles Nd6793, Sbl903, Gsp8241 and Gsp8239. 61 However, weak bootstrap supports (below 70%) were observed in the following clusterings: the subgrouping o f five het-cGR - type alleles, the lineage separation o f the ten het-cPA - type alleles and the eleven het-cOR - type alleles (Figure 2-9). Nonetheless, the subgrouping of the nine het-cPA - type alleles from four species was supported by 87% of the bootstrap replicates (Figure 2-9). Most of the het-c allele groups were also observed in the tree reconstructed using maximum likelihood (Figure 2-11). The data indicate that the subgroupings of het-c by allelic types were not significantly affected by the D N A sequence alignments. These analyses suggest that the D N A sequences o f het-c specificity region display trans-specific polymorphism; alleles from the same allelic types are more similar than alleles from different allelic types but from the same species. For example, in all the trees, a het-cPA - type allele from N. crassa (Nc4832) is more closely related to another het-cPA - type allele from TV. intermedia (M3290) than to a het-cOR - type allele from N. crassa (Nc847). A n interesting finding is that the trans-specific polymorphism of het-c is also apparent within each allelic group. For instance, in 500 neighbor-joining trees constructed using D N A sequences from only het-cPA - type alleles (Figure 2-12), 97% o f the bootstrap replicates supported the trans-specific clustering of Nc4832, Ni2316 and Ni3290, and the subgrouping ofNt6583, Nc4711, Nci 130, Nt 1270, Ndi3228 and Nc4486; all o f the replicates supported the clustering of Gsp 8243, Np7221 and Ss2740. Nearly identical clustering patterns were observed in trees constructed using maximum likelihood method (data not shown). Trans-specific lineages are also observed within both het-cOR - type alleles and het-cGR - type alleles. In 500 neighbor-joining trees of het-c0R - type alleles (Figure 2-13), 82% of the bootstrap replicates supported the clustering o f TV. crassa allele Nci824 with N. intermedia 62 allele Ni3721, and the subgrouping ofNdi6793, Sbl903, Gsp8241 and Gsp8239; 84% of the bootstrap replicates supported the clustering of alleles Nc4481 and Gsp8242. In 500 neighbor-joining trees o f het-c - type alleles (Figure 2-14), all the bootstrap replicates (100%) supported the subgrouping of N. crassa allele Nci945 with N. discreta allele Ndi6788 and the subgrouping of Nc967, Ss2741, Sb7140 and Ndol692. 63 88 85 100 80 r - t Nc2489 O Nc1693 0 Nc4709 O ,_|' Nc847 O Nc4481 O Gsp8242O Sh2739 O NH940 O NU 799 O Ns963 O Ns5940 O 97 93 - Ndi3228 P - Nc4486P - Nc4711 P Nc1130 P NH270P Nt6583 P Ni2316P NI3290 P 100 rt Nc4832 P Ns5941 P Sb1903 O — Ndi6793 O Gsp8239 O Gsp8241 O OR 95 Ss2740P - Np7221 P OR PA PA Ndo1692G Nc1455G Nc4499 G 94 Nc967G — Sb7140 G SS2741 G NC1945G — Ndi6788 G Gsp8243P GR •c Nc1824 0 Ni3721 O OR Ndi5923 X 0.1 nucleotide substitution per site Figure 2-6. Unrooted neighbor-joining tree from the D N A sequence (390 nucleotides) alignment in the het-c specificity region including the insertion/deletion sites. Bootstrap support is shown by percentage out of 500 replicates. Percentages below 75% are not indicated. Branch lengths are proportional to the genetic distances. The designation OR, het-c0R allelic-type; Yh-het-cPA allelic type; and GR-het-cGR allelic type is based on the alignment in Figure 2-5 and is used in all the following Figures. 64 99 Nc2489O Nc4709 O Nc847O Nc1693 O Nc4481 O — Gsp8242O I Ns963 O *Ns5940O Sh2739 0 OR |_| NH940 O NH 799 O 100 rt SM903 O Ndi6793 O Gsp8239O Gsp8241 O I Nc4486 P Nt1270P Nd 130 P f- Ndi3228 P r- Ni3290 P U - Nc4832 P ^ Ni2316P A/c47^ P Nt6583 P - Ns5941 P 86 94 SS2740P - Np7221 P PA Gsp8243P Ndo1692 G | GR 92 Nc1455 G — Nc4499 G Nc967G Sb7140G Ss2741 G •c NC1945G — Ndi6788 G GR • Nc1824 O Ni3721 O Ndi5923X I OR 0.1 nucleotide substitution per site Figure 2-7. Unrooted neighbor-joining tree from the D N A sequence (327 nucleotides) alignment in the het-c specificity region excluding the insertion/deletion sites. Bootstrap support is shown by percentage out of 500 replicates. Branch lengths are proportional to the genetic distances. 65 85 100 97 87 100 " Nc4481 O • Gsp8242 O Nc4709 O Nc2489 O Nc1693 0 Nc847O Sh2739 O NH940 O NH799 0 OR Ns5940 O Ns963 O 100 Gsp8239 0 Gsp8241 O Sb1903 O Ndi6793 O Nci824 O Ni3721 O Nc1945G Ndi6788 G Nc1455G Nc4499 G — Nc967G Ss2741 G Sb7140G GR Ndo1692G 96 99 92 NU270P Nd 130 P Nc4711 P Ndi3228 P Nc4486P Nt6583 P Ni2316P Ni3290 P Nc4832P 99 Ns5941 P Ss2740P Np7221 P Gsp8243P PA Ndi5923X Figure 2-8. Unrooted parsimony tree from the D N A sequence (390 nucleotides) alignment in the het-c specificity region including the insertion/deletion sites. Bootstrap support is shown by percentage out of 500 replicates. 66 51* 98 50* Nc2489 O Nc4709 O Nc847 O Nc1693 O — Gsp8242 O N&4481 O Ns963 O Ns5940 O Sh2739 O Nil940 O NH799 O OR 84 87 Nt1270P Nc1130 P NC4711 P - Nc4486 P - Ndi3228 P Ni3290 P Ni2316P PA NC4832 P Nt6583 P Ns5941 P 99 85 Sb1903O Ndi6793 O SS2740 P Np7221 P Gsp8241 O Gsp8239O PA OR Gsp8243P 56" Ndo1692 G Nc4499 G Nc1455 G - Nc967 G Sb7140 G Ss2741 G - NC1945G - Ndi6788 G Ni3721 O Nc1824 I GR GR o I OR Ndi5923 X Figure 2-9. Unrooted parsimony tree from the D N A sequence (327 nucleotides) alignment in the het-c specificity region excluding the insertion/deletion sites. Bootstrap support is shown by percentage out o f 500 replicates. 67 Ndi5923X — Sh2739 O Gsp8242O Nc4481 O Nc4709 O Nc2489 O Nc847O '— Nc1693 0 I— NH940 O NU 799 O Ns5940 O Ns963 O I— Ndi6793 O SM903 O — Gsp8241 O Gsp8239O - Sb7140 G Ss2741 G Nc967G - | Nc4499 G L p Nc1455 G Nc1945G Ndi6788 G Ni3721 O Nc1824 0 Gsp8243 P GR OR Np7221 P SS2740 P Ndo1692G | GR PA OR Ns5941 P NC1130P Nt1270P K Nc4486P h Ndi3228 P M6583 P PA r Ni \ Nc4711 P c4832 P Ni3290 P Ni2316P OR Figure 2-10. Unrooted maximum likelihood tree from the D N A sequence (390 nucleotides) alignment in the het-c specificity region including the insertion/deletion sites. 6 8 Ndi5923X rL' NH799 O NH940 O Sh2739 O Nc4709 O Nc2489 O Nc847 O Nc1693 0 Gsp8242 O Nc4481 O - N  i3721 O Nc1824 O Ndi6788 G Ns5940 O Ns963 O I— Nd6793 O SM903O Gsp8241 O Gsp8239 0 OR Nc1945 G Sb7140 G SS2741 G Nc967 G • Nc4499 G Nc1455 G GR Gsp8243P Np7221 P SS2740P Ndo1692G |GR PA OR NS5941 P - Ni3290 P - Nc4832 P - Ni2316P - Ndi3228P L M1270P NC1130P — Nc4486 P j M6583 P ' Nc4711 P OR PA Figure 2-11. Unrooted maximum likelihood tree from the D N A sequence (327 nucleotides) alignment in the het-c specificity region excluding the insertion/deletion sites. 6 9 Ndi5923X 100 Ns5941 P 97 94 Nt6583 P Nc4711 P NC1130P NH270P Ndi3228 P Nc4486 P Nc4832P Ni2316P Ni3290 P 100 Gsp8243P Np7221 P Ss2740 P Figure 2-12. Unrooted neighbor-joining tree of het-c - type alleles. Bootstrap support for a clade is shown as the percentage of 500 replicates containing the clade. Percentages below 75% are not indicated. The sequence length is 375 nucleotides. 70 82 84 100 100 C c Nc84 7 0 Ncl693 O Nc4709 O Nc2489 O SH2739 O Ns5940 O Ns963 O Ncl824 O N13721 O Nd6793 O 100 93 Sbl903 O Gsp8241 O Gsp8239 O 9 6 I Nil 940 O Nil 799 O Nc4481 O Gsp8242 O Figure 2-13. Unrooted neighbor-joining tree of het-c - type alleles. Bootstrap support for a clade is shown as a percentage o f 500 replicates. Percentages below 75% are not shown. Note that neither species nor genera are monophyletic. The sequence length is 345 nucleotides. 71 Ndi6788 G Nc1945G 100 Nc967G 64 Ss2741 G Sb7140 G Ndo1692G Nc4499 G Nc1455 G Figure 2-14. Unrooted neighbor-joining tree o f het-c - type alleles. A n N. crassa allele Nc967 is more closely related to a S. sclerogenia allele Ss2741 than to N. crassa alleles Nc4499 or Nci455. Bootstrap is shown as a percentage o f 500 replicates (only > 60% are shown). Again, alleles from the same species or genera are not monophyletic. The sequence used is 327 nucleotides. 72 2.3.5 Numbers of synonymous and nonsynonymous substitutions per site in the het-c specificity region. Theoretical studies predict that positive Darwinian selection should enhance the number of nonsynonymous nucleotide substitutions (Lopez de Castro et al., 1982). To test i f positive selection was acting in favor of polymorphisms in the het-c specificity domain, the number o f nonsynonymous substitutions per nonsynonymous site (d^) and synonymous substitutions per synonymous site (ds) in the polymorphic (filled bar region in Figure 2-15, as highlighted in Figure 2-5, total 57 codons) and non-polymorphic (clear region in Figure 2-15, total 73 codons) region of 130 codons was examined. Histograms o f how the per-site rates o f change mapped onto the codons for both synonymous and nonsynonymous sunstitutions are shown in Figure 2-15. In all 40 het-c alleles, the ratio of dj/ds in the polymorphic region (20.4 / 38 = 0.54) significantly exceeded that in the non-polymorphic region (2.6 / 24.5 = 0.11; z test, p < 0.005; Table 2-3) due to the significantly increased value o f dN in the polymorphic region. A significantly increased d^/ds ratio in the polymorphic region was also observed in the separate analyses of het-c alleles from het-cOR - type, het-cPA - type and het-cGR - type, N. crassa isolates and the genus Sordaria (Table 2-2). The most variable alleles contained the het-cPA - type insertion, where the dy ds ratio reached 1.3 in the polymorphic region. In the estimation of d^and ds among all het-c alleles, the ds value was generally higher than the value o f dN with the exception of the polymorphic region of PA-type alleles (Table 2-2). However, the value of dN was significantly higher in the polymorphic region than that in the non-polymorphic region in all analyses (Table 2-2, z test, p < 0.005) while the ds values in the two regions showed no significant differences (z test, p < 0.05). Thus, the 73 ratio o f dj/ds in the polymorphic region was significantly greater than that of the non-polymorphic region (Table 2-2). 74 1.00 0.75 H 0.50 0.25 0.00 a) r W f r 0 20 40 60 80 100 120 Codon position 2.0 1.5 H d 1 0 0.5 H 0.0 b) i l i i 41 0 20 40 60 80 100 120 Codon position Figure 2-15. a) The average number of nonsynonymous nucleotide substitutions per nonsynonymous site and b) the average number of synonymous nucleotide substitutions per synonymous site grouped by codon of het-c specificity domain and its flanking region. The filled bar region represents the polymorphic region of het-c as highlighted in Figure 2-5. Clear bar region represents the flanking region. Codon position 40 corresponds to the position of the first amino acid residue in Figure 2-5. The 30 amino acids from codon 40 to 60 and from codon 120 to 130 are identical in all alleles. The numbers o f nonsynonymous and synonymous substitutions were calculated by M E G A (Kumar et al., 1993). A l l 40 alleles were used in the analysis. Note the difference in ordinate scales used for d N and ds. 75 Table 2-2. Comparison of number of synonymous (ds) and nonsynonymous (dn) nucleotide substitutions per 100 sites in het-c polymorphic (PM) and non-polymorphic (Non-PM) region. Alleles/ Species P M Region N o n - P M Region d s d N d s d N 38.0 20.4 24.5 2.6 (7.1) (2.8) (3.5) (0.6) OR-type* 25.5 10.3 19.2 2.1 (5.6) (1.7) (3.5) (0.6) GR-type* 16.2 6.0 14.0 1.4 (5.1) (1.5) (3.3) (0.6) PA-type*** 16.3 21.5 13.8 1.2 (4.5) (2.9) (3.0) (0.4) N. crassa** 23.9 17.0 20.5 2.1 (7.8) (3.3) (4.3) (0.7) Sordaria**' 37.0 20.3 27.3 1.5 (8.9) (3.8) (5.5) (0.7) Polymorphic (PM) and non-polymorphic (non-PM) regions refer to the codons of filled (PM) and clear (non-PM) bar regions in Figure 2-5, respectively. N gives the number o f alleles surveyed. Standard errors are in brackets. OR, G R and PA-type refer to het-c0R, het-cGR and het-cPA allelic specificity types, respectively. Test o f hypothesis: d^ds ratio has no significant difference in the two regions. * P < 0.05, ** P < 0.01, *** P < 0.001. 76 2.4 D I S C U S S I O N 2.4.1 Trans-specific polymorphisms at het-c Three methods were used for het-c phylogenetic inference from D N A sequence data. Trans-specific polymorphisms were observed for het-c alleles using all three methods. Regardless of whether the insertion/deletion motifs were included or excluded, alleles clustered according to allelic types rather than according to species. In cases of including and excluding the insertion/deletion sites in the D N A sequences, maximum likelihood analysis resolved nearly identical subgroups according to het-c allelic types (Figure 2-10 and Figure 2-11); the neighbor-joining also gave similar subgroupings with similar robustness of bootstrap support (Figure 2-6 and Figure 2-7). Most o f the subgroups in parsimony trees were similar with strong bootstrap support regardless of whether the insertion/deletion motifs were included or excluded (Figure 2-8 and Figure 2-9). The het-c region that confers allelic specificity displayed a high degree of polymorphism in D N A sequences. Thirty-nine het-c alleles were identified in the 40 Sordariaceae sequences examined and fifteen were identified in N. crassa alone. In the phylogenetic analyses o f the 39 alleles, het-c formed lineages according to allelic types rather than species, suggesting that balancing selection is operating to maintain the het-c polymorphisms. Interestingly, the trans-specific polymorphisms are not limited to the three het-c allelic types, defined as het-c0R - type, het-cGR - type and het-cPA - type, but are also observed within an allelic type (Figure 2-12 - 2-14). Trans-specific polymorphisms have been well demonstrated at the MHC and the S loci, both o f which are believed to be under balancing selection and function as a self and 77 non-self recognition system. A t the MHC loci , certain HLA alleles are more similar in their sequence to certain chimpanzee MHC alleles than to other HLA alleles (Lawlor et al., 1988; Mayer et al., 1988). A t the S-loci, the trans-specific pattern of polymorphism has been demonstrated in both gametophytic and sporophytic self-incompatible systems (Dwyer et al., 1991; Ioerger et al., 1990; Richman et al., 1996). Trans-specific polymorphism is not restricted to loci under balancing selection, such as the MHC loci and the S loci , but by the persistence of neutral polymorphisms i f speciation times are short (in terms of generations) and i f the effective population size is too large (Nagal et al., 1998). A well studied example of neutral trans-specific polymorphisms is represented by genes in the haplochromine species flock in Lake Victoria that underwent rapid diversification. In Lake Victoria cichlid fish, neutral trans-specific polymorphisms have been detected in the non-coding sequences of 3 ' U T R or introns of randomly selected nuclear genes and also in association with structural genes under purifying selection (e. g., G6P, a gene encoding glucose-6-phosphatase) (Ngal et al., 1998; reviewed in K l e i n et al., 1998). Most of the polymorphisms were estimated to have arisen more than 12,000 and less than two mil l ion years ago. It was proposed that the neutral polymorphisms in Lake Victoria species were passed on to the descendent species as they emerged during the explosive diversification and speciation events that created the extant flock (reviewed by Kle in , 1998). However, the Victoria cichlid fish represents an extreme case o f rapid speciation, where the maintenance of polymorphisms across speciation events maybe expected. The divergence among the Sordariaceae examined here are thought to be much older (Berbee and Taylor, 1992;Berbeeetal. , 1995). 78 2.4.2 het-c allelic lineages were generated prior to the divergence of modern species Sequence comparison and phylogenetic analysis o f the het-c specificity region revealed the following associations: 1) the het-c sequences are highly divergent between allelic types and between alleles within each allelic type (Appendix 7.1); 2) het-c shows ubiquitous trans-specific polymorphism between allelic types and within allelic types (Figure 2-6 to Figure 2-14); 3) allele lineages were generated before the divergence o f Neurospora and Sordaria and persisted through multiple speciation events. The divergence time of Neurospora and Sordaria was calculated according to the 18S rRNA sequences, for which the molecular clock has been calibrated using fossil evidence and ages o f fungal host and symbionts (Berbee et al., 1995). The D N A sequences of the 18S rRNA genes from Sordaria fimicola (Genebank access No . X69851) and N crassa (Berbee and Taylor, 1992) show a 1.46% difference, indicating a 0.73% substitution per lineage. The calibrated nucleotide substitution rate is 0.5 - 2% per 100 mil l ion years in fungi (Berbee et al., 1995). Thus, Sordaria diverged from Neurospora at least 36 mil l ion years ago, indicating that the het-c polymorphism has been retained for at least this time interval. Long persistence of allelic lineages was also observed in both the MHC and S loci . A t the DRB1 locus, allelic lineages have been retained for at least 30 mil l ion years (Ayala et al., 1994), while the oldest allelic lineages diverged approximately 55 mil l ion years ago (Martin, 1993; Satta et al., 1996a, 1996b). A t the S loci , allelic lineages have been retained for at least 36 mil l ion years, while the oldest allelic lineages diverged more than 70 mil l ion years ago (Ioerger et al., 1990). The persistence of polymorphism within the het-c specificity domain within the different genera during such a long period of time strongly implies that allelic variants have been maintained by balancing selection across multiple speciation events. 79 2.4.3 het-c allelic lineages OR Although only three allelic specificities have been identified in TV. crassa (het-c , het-cGR and het-cPA), at least seven distinctive allelic lineages were resolved in most of the het-c phylogenetic trees - three het-cOR lineages, two het-cPA lineages and two het-cGR OR lineages. In all the neighbor-joining trees and maximum likelihood trees, two het-c - type alleles, Ni3721 and Nci824, were included in the het-cGR lineage. The clustering o f these two OR-type alleles with GR-type alleles was probably due to the affect o f the intragenic (interallelic) recombination event involving the polymorphic block M E E R G G H / I H E K E T R S (Figure 2-5). Similarly, Gsp8243, Np7221, and Ss2740 were separated from the PA-lineage probably because of recombination between the polymorphic block I, M E E R R G G H / I H ( Y ) K K ( N ) E T G E ( R ) . The inclusion of Ndol692 in the PA-lineage (Gsp8243, Np7221, and Ss2740) is also due to the effect of the polymorphic block I. The formation of a separate OR-lineage that is made of Sb1903, Ndi6793, Gsp8239 and Gsp8241 is due instead to sequence variation in polymorphic block I and II in these alleles (Figure 2-5, Appendix 7.1). Three factors could explain the occurrence o f seven het-c allelic lineages. First, gradual accumulation of spontaneous nucleotide substitutions resulted in allelic lineages that arose at different evolutionary time intervals. Second, segmental sequence exchanges could have occurred between two lineages through interallelic (lineage) recombination. Consistent GR OR with this explanation, apparent mosaic sequences between het-c - type and het-c - type, plus het-cGR - type and het-cPA - type alleles were observed. For instance, N13721 and N c i 8 2 4 are chimeras between H E T - c G R (the polymorphic block IH(Y)EKETG(R)S(E) ) and H E T - c 0 R (the OR-specific pattern of insertion R N D T R ) ; Np7221, Gsp8243, Ss2740 and Ndo l692 are chimeras between H E T - c G R (the polymorphic block 80 fflEKETGS/IYE(K)NEN(T)PRR) and H E T - c (PA-specific pattern o f insertion). Under this hypothesis, the polymorphic block in het-c would have a strong effect on the placement of these chimeric alleles in phylogenetic tree reconstructions, and thus additional het-c allelic lineages were formed. However, all lineages showed a trans-specific pattern, suggesting that generation o f these exchanges was in a common ancestor o f these or that recombination has happened more than once. Intragenic (interallelic) segmental exchanges were also observed in Feline MHC class I D R B genes based on the nature o f a highly mosaic structure among the D N A sequences (Yuhki and O'Brien, 1990; 1994). Third, selection was acting on the functional region of H E T - c in relationship to its role for non-self recognition. Negative (purifying) selection acted to conserve certain residues in regions required for common H E T -c function; positive selection acted to produce sequence variations in regions that are important for H E T - c specificity. I f this is true, do the het-c allelic lineages in the phylogenetic analyses represent different functional het-c lineages that confer alternative specificities? It is known that the three het-c allelic types, het-cOR - type, het-cPA - type and het-cGR - type are mutually incompatible in natural heterokaryons (Garnjobst, 1953; Mylyk , 1975; Perkins, 1988), laboratory genetic analyses (Saupe and Glass, 1997) and transformation assays in N. crassa (Chapter 3). However, only four het-c specificities were detected among the 39 alleles (allele Ndi5923 confers different specificity from het-cOR, het-cPA and het-cGR) by chimeric construction and transformation assay (Chapter 3). The fact that different phylogenetic lineages were observed within a het-c allelic - type permits the following speculation: strains carrying the same allelic - type o f het-c but assigned in different lineages may be incompatible in nature, but laboratory assays are not discriminating enough to differentiate them. 81 2.4.4 Possible mechanisms of balancing selection at the het-c locus Balanced allelic polymorphisms can be maintained either by over-dominant selection (heterozygotes have a superior fitness) in a diploid organism or by frequency-dependent selection (rare alleles have a selective advantage) (Takahata and Ne i , 1990; Takahata et al., 1992). Both selection mechanisms have been proposed to occur in the MHC and the S loci for the maintenance of polymorphisms (for reviews, see Hughes and Yeager, 1998; Charlesworth and Awadalla, 1998; and also see Introduction). In haploid filamentous fungi, heterozygote superiority (overdominance) selection is an unlikely force for the persistence o f het-c polymorphisms. However, the possibility of overdominant selection at het-c can not be ruled out until het-c is shown to be inactive in the transient sexual dikaryon phase. In a population analysis o f 36 Neurospora crassa isolates from a 5-hectare sugar cane field of Lousinana, the three het-c allelic - types showed nearly equal frequency o f occurrence (this study and Louise Wheeler, personal communication). In a restriction fragment length polymorphism (RFLP) analysis of the P C R products of the het-c specificity region from the 36 isolates, fourteen showed a het-cPA pattern, fourteen had a het-cOR pattern and eight had a het-cGR pattern. When genomic D N A from the 36 isolates was digested and probed with a 35-kbp cosmid containing het-cOR, twenty-four different R F L P patterns were observed (Louise Wheeler, personal communication) indicating that the population was not clonal. These studies suggest that maintenance of het-c polymorphism within fungal populations is possibly attributable to frequency-dependent selection. 82 2.4.5 An excess of nonsynonymous nucleotide substitutions in the het-c polymorphic region A powerful method of discriminating between selection and neutral polymorphism is to compare the number of synonymous and nonsynonymous nucleotide substitutions per site (Hughes and Ne i , 1988, 1989). In the case of positive selection favoring diversity at the amino acid level, the nonsynonymous substitution rate is enhanced (Hughes and Ne i , 1988). Under purifying selection, as occurs in the case of most structural protein-coding genes or functional constraint region of some other molecules, the rate of synonymous substitution is higher (Kimura, 1983). Hughes and N e i (1988) hypothesized that natural selection is responsible for the increased nonsynonymous nucleotide substitution rates. Significantly enhanced nonsynonymous nucleotide substitution has been shown in the hypervariable regions of both the MHC molecule and S alleles. It was found that the nonsynonymous nucleotide substitution was significantly enhanced in the 57 codons encoding the P B R of the MHC class I molecules (Nei and Gojobori, 1986; Cadavid et al., 1997). A similar pattern was also found in the putative P B R in the class II MHC genes (Hughes and N e i , 1989; Y u h k i and O'Brien, 1997; Mi l l e r and Withler, 1996). A t the S loci , higher numbers of nonsynonymous nucleotide substitutions were observed in the receptor domains o f SRK (S-Receptor Kinase) and SLG (S-Locus Glycoprotein) genes in the Brassiceae (Hinata et al., 1995) and the hypervariable regions of S alleles in the Solanaceae (Clark and Kao, 1991; Ioerger et al., 1990). The hypervariable regions of these molecules play a direct role in the recognition of self and non-self and are believed to be under balancing selection to maintain allelic polymorphisms 83 (Hughes and Yeager, 1998; K l e i n et al., 1998; Charlesworth and Awadalla, 1998; Seibert et al., 1995). To assess the effect that selection has on the pattern o f nucleotide substitution in het-c, the number and frequency of synonymous and nonsynonymous nucleotide substitutions inside and outside of the het-c variable region were compared. In the hypervariable region o f het-c, both the numbers and frequency o f nonsynonymous nucleotide substitutions were significantly higher. Since the number o f synonymous nucleotide substitutions per site (ds) is expected to reflect the neutral mutation rate (the fraction of neutral mutations at synonymous sites being close to 100%, Kumar et al, 1993), the nearly uniform ds values in H E T - c indicate nearly-equal neutral mutation rates inside and outside the H E T - c polymorphic regions. Therefore, the enhanced number o f nonsynonymous nucleotide substitutions (dx) in the polymorphic region is unlikely due to a higher mutation rate at these sites. Examination o f the pattern of nucleotide substitutions in the het-c specificity region indicates that positive selection has acted to enhance nonsynonymous nucleotide substitutions favoring the diversity of het-c at the amino acid level. This evidence supports the hypothesis that H E T - c has a selectively important function functions for non-self recognition, as with the M H C molecule and the S protein. 2.4.6 The het-c locus shares evolutionary features with MHC and S loci In higher eukaryotes, the loci in the MHC and the S system are examples for which balancing selection has caused the retention o f ancestral polymorphisms - trans-specific polymorphism. Both the MHC and the S loci have a large number o f alleles present within a single species. In human MHC (the HLA complex) at the most polymorphic class I (HLA-B) 84 and II (HLA-DRB1) loci , 150 and 156 alleles have been identified, respectively (Bodmer et al., 1997). A t the S locus, 30 to 50 S-alleles are commonly found per population in the Solanaceae (Lawrence et al., 1993) and more than 100 5-alleles have been estimated in Brassica campestris (Nou et al., 1993). Due to the lack of a large number of Neurospora isolates from a defined population, it has been difficult to estimate the number of existing het-c alleles in natural populations (Glass, personal communication). However, 39 het-c alleles have been identified in the Sordariaceae and at least seven allelic lineages were resolved in the phylogenetic analyses. The data in this study demonstrate that the het-c region conferring specificity shares several common evolutionary features with the MHC and S loci . First, alleles display large divergences in D N A sequence. Second, trans-specific polymorphisms are apparent. Third, nonsynonymous nucleotide substitutions are enhanced within the specificity region. Fourth, allelic lineages persist for a long period of time. These data suggest that het-c is subject to balancing selection. This study is consistent with the hypothesis that het-c acts as a locus important for non-self recognition in the Sodariaceae and that vegetative incompatibility is a bona fide non-self recognition system in filamentous fungi that limits heterokaryon formation between unlike individuals. Moreover, comparable evidence for the persistence of ancient polymorphisms at the het-c locus, mammalian MHC loci and plant S loci reinforces the view that balancing selection is an evolutionary mechanism common to different non-self recognition systems. 85 C h a p t e r 3 M o l e c u l a r basis o f het-c al lele speci f ic i ty 3.1 I N T R O D U C T I O N A s was described in the general introduction, the het-c locus encodes three allelic specificities termed het-cOR, het-cGR and het-cPA based on genetic studies (Saupe and Glass, 1997). Polypeptides encoded by the three alleles show 86% amino acid identity and differ by variable domains. B y chimeric construction between the three het-c alleles, a 34 - 48 amino acid variable region that is dissimilar in H E T - c 0 R , H E T - c G R and H E T - c P A was determined to confer allelic specificity (Saupe and Glass, 1997; Figure 1-7 and Figure 1-8). The variable domain of H E T - c 0 R , H E T - c G R and H E T - c P A differs in both amino acid sequences and the pattern of insertion/deletion. A n examination of this region in related species and genera to TV. crassa in Chapter 2 showed that all 40 alleles surveyed fell into one of the three het-c allelic types based on the pattern o f insertion/deletion within the het-c specificity region (Figure 1-8; W u et al., 1998). However, within each of H E T - c 0 R , H E T - c G R and H E T - c P A allelic types, amino acid sequences in the variable region was highly diversified among alleles. In this chapter, I address the question of whether the amino acid sequence of a particular het-c specificity domain or the pattern of insertion/deletion is the primary factor for determining specificity. To differentiate these two possibilities, I take advantage of the natural amino acid variation and deletion/insertion motifs among the 39 unique alleles isolated from different genera and species described in Chapter 2. First, I addressed whether the het-c specificity of these alleles, as assayed in N. crassa, was affected either by amino 86 acid sequence and/or insertion/deletion pattern in the specificity domain. B y chimeric construction and transformation assays, I observed that the pattern o f insertion/deletion in the specificity domain is the most critical determinant for het-c allele specificity. Second, I further investigated how the pattern o f insertion/deletion affects het-c specificity by construction of a number of artificial het-c alleles using P C R mutagenesis. The combination of amino acid sequences and insertion/deletion motifs of these artificial alleles was not observed in the survey o f natural alleles. B y this method, I was able to generate a number of novel het-c specificities. The mechanism of allele specificity has been examined in several fungal non-self recognition systems by the construction of chimeric (or hybrid) alleles. In P. anserina, a single amino acid difference in the proteins encoded by the vegetative incompatibility locus, the het-s locus, was sufficient to confer allele specificity (Deleu et al., 1993). Such a situation was also observed at the het-c locus in P. anserina (Saupe et al., 1995). In Ustilago maydis, a region composed of 30 to 48 amino acid residues was identified that regulates allele specificity at the b mating locus (Yee and Kronstad, 1993), and artificial hybrid b alleles with novel specificity were generated by chimeric construction within this border region (Yee and Kronstad, 1998). In Coprinus cinereus, specificity of the homeodomain mating protein HD1 and H D 2 was determined by the N-terminal 160 to 170 amino acids (Banham et al., 1995). In the case of the homeodomain mating proteins in Schizophyllum commune, named the Y and Z proteins, a region of 55 amino acids in the Y protein and 41 amino acids in the Z protein was defined by chimeric allele construction that determined the specificity (Wu et al., 1996). Overall, these studies defined important elements involved in specificity determination and mediating non-self recognition. 87 3.2 MATERIALS AND MATHODS 3.2.1 Strains and media A list o f N. crassa strains used for transformation and heterokaryon tests is given in Table 3-1 and Table 3-2, respectively, along with their relevant genotypes. A l l strains were maintained on Vogel's media (Vogel, 1964) with required supplements. Escherichia coli strain DH5a (F-, e n d A l , hsdRJ7, supE44, lacZM15; Bethesda Research Laboratory) was used for routine D N A manipulation work and was grown in L B medium (Sambrook et al., 1989). Table 3-1. List of A. crassa strains used as transformation recipients Strain designation Genotypes Origin C2-2-9 het-6PA het-cOR thr-2 A M . L . Smith C9-2 het-6°R het-cPA thr-2 a M . L . Smith F G S C 2193 NM149 T(II;V), het-6°Rhet-cGR A D . D . Perkins C 1 5 - l a het-6°R het-cOR ; pan-2; arg-5; inl a R. Todd Xa-3 het-60R het-cPA; pan-2; arg-5 A Q. Xiang X22-2 het-6°R het-cnul1 thr-2 a Q. Xiang CJ44 het-6°R het-cnu"; pan-2; arg-5 A This study 88 Table 3-2. List of N. crassa strains used for heterokaryon tests. Strain designation Relevant genotypes Origin 6-13 ad3B arg-1; het-6PA het-cPA pyr-4 A C . Y a n g RLM58-18 het-6PA het-cPA pyr-4; inl a R. L . Metzenberg C8c l58 ro-7 un-24 het-6°R het-cPApyr-4 A M . L . Smith C3cr-29 ro-7 un-24 het-6°R het-cPA pyr-4; inl a M . L . Smith 6-28 ad3B arg-1; het-6PA het-cOR; inl A C. Yang 6-19 ad3B arg-1; het-6PA het-cOR pyr-4; inl a C. Yang FGSC4571 ad3A; un-24 het-6°R het-cOR; nic-2; cyh-1 Am54 A . J. F . Griffiths 3.2.2 Recombinant DNA techniques Standard protocols were used for D N A manipulations (Sambrook et al., 1989). Restriction and D N A modifying enzymes were obtained from Bethesda Research Laboratories, Boehringer Mannheim, or New England Biolabs and were used according to the manufacturer's instructions. P C R was performed with Perkin-Elmer Cetus D N A Thermal Cycler. The pCRIIvector (InVitrogen, San Diego, C A ) was used for cloning P C R products. Q I A E X I I gel extraction kit (Qiagen Inc, Mississauga, Ontario) was used for recovering D N A fragments from agarose gels. 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EE! EE! £ § P H H i H i H1 > > > H i H i H i W W W CO CJ CJ £ £ £ CO CO CO 3 3 3 Pi Pi Pi fa fa fa P P P W W W Hi Hi Hi P i W H i H i EH EH & >, Pi Pi H EH e e CD CD Pi PI fafa Pi PI CO CO I I I I X X O CD CD CD a a w w §1 p p H H H1 H1 > > Hi H i w w CJ CJ CO CO 3 3 Pi PI fa fa P P W W H1 H1 I CN O S cn •<H 0\ CO <J\ CN tv O C\ 00 Vo 0>> CS H ' H H IN fe; fe; co co H 0> CS) rn MI o o\ CN CN CN V 0>i 00 00 CO OS CN D i D i Q | i o M to i f i f « ' H CD CD CD fe; fe; 90 Figure 3-1. Amino acid sequences o f the specificity region from naturally occurring het-c alleles. Region underlined is variable. Asterisks indicate conserved sites. Dash represents deletion motifs. Regions I and II are polymorphic blocks. P A (Panama), O R (Oakridge), G R (Groveland) refer to the three genetically characterized alleles that confer different het-c specificity (Saupe and Glass, 1997). 91 3.2.3 Isolation of naturally occurring het-c alleles The specificity region of natural het-c alleles was obtained by P C R amplification of genomic D N A from isolates described in Chapter 2 using Red and Ye l low primers (Chapter 2, section 2.2.3). A l l the P C R products were cloned into pCRII vector for further manipulations. Inferred amino acid sequences of the specificity region of the natural alleles used in this study are shown in Figure 3-1. 3.2.4 Generation of artificial het-c alleles Artificially constructed het-c alleles were generated using recombinant P C R techniques (Horton et al., 1989; Vallette et al., 1989). The technique requires two sets of primers and two rounds of P C R . The principles are diagrammed in Figure 3-2 and Figure 3-3, which represent the generation of recombinant molecules and in-frame deletion/insertions, respectively. The oligonucleotides used in the construction are listed in Table 3-3. Primer sets Red and Spl .3 were used in the second round P C R reaction for all allele constructions. Specific primer sets and templates for generating specific alleles in the first round P C R are shown in Table 3-4. Oligonucleotide primers were synthesized by the U B C N A P S unit (Biotechnology Laboratory). A l l P C R reagents were supplied by Boheringer Mannheim. Plasmids containing het-c clones were used as first round P C R templates. The first round P C R was performed by the standard protocol: initial 5 min denaturation at 94°C, followed by 30 step cycles of 1 min at 94°C, 1 min at 55 °C and 1 min at 72°C, and a final extension at 72°C for 10 min. Reactions were carried out in volumes of 50 ul containing 10 m M T r i s - H C l p H 8.3, 50 m M KC1, 2.0 m M M g C l 2 , 25 u M each of d A T P , dCTP, dGTP, dTTP, 0.2 u M of each primer, 1.25 92 unit of Taq polymerase and 50 ng of template plasmid D N A . The first round P C R products were purified from an agarose gel using the Q I A E X I I Ge l Purifying kit. Two corresponding first round P C R products were combined and used as templates for the second round P C R to construct recombinant products. The second round P C R conditions were similar to that of the first round except that annealing was done at 51°C and an auto extension for 10 seconds at 72° C was performed after each cycle. P C R products from the second round were cloned into pCRII vector (InVitrogen, San Diego, C A ) for further manipulations. 93 Figure 3-2. P C R generation of recombinant het-c alleles. The procedure is diagrammed for the construction of the pol allele that has both het-cOR - specific and het-cPA - specific insertion motifs; the same strategy was used to construct the rest of the recombinant het-c alleles. The oligonucleotides P01.5 and P01.3 were designed to have overlapping ends. The red and spl.3 primers were used for the second round P C R for generation o f all artificial het-c alleles. 94 Figure 3-3. P C R generation of het-c alleles by deletion. The procedure is diagrammed for the construction of pdn alleles (pdl, pd2, pd3, pd4), which have a different size of insertion in the het-cPA - specific motif; the same strategy was used to construct alleles deli and odl. Allele del3 was generated from het-c with the deletion of the three residues, N N G . Allele odl was generated from het-cOR with the deletion of five residues E N D T R from het-cOR specific motif. The oligonucleotides Pdn. 5 and Pdn.3 were designed to have overlapping ends. The Red and Spl.3 primers were used for the second round P C R for generation of all artificial het-c alleles. 9 5 Table 3-3. Oligonucleotide primers for the construction of artificial het-c alleles. Primer name Sequence3 Red G G A G A C A T G G C G A T A T C G Spl .3 G C T C A T G C C A G G A A C A A C Del3.3 A G C G T A C T T G A G T T T G A G T T T A G T C G C A G T G C Del3.5 A C T C A A A C T C A A G T A C G C T T G G C C C C T G G T P01.3 C T G A A C T G T C C A T T G T T A C G T G T G T C A P01.5 C A C G T A A C A A T G G A C A G T T C A G G A G C G T A A G A P02.3 G T C T A T T C T C A C G T G T G T C A T T C T T G A G P02.5 G A C A C A C G T G A G A A T A G A C A G T T C A G G C G C G T A A G A P03.3 C T G T C T A T T C T C G A G T G T G A T T C G C G T A T C P03.5 C A C A C T C G A G A A T A G A C A G T T C A G G C G C G T A A G A P04.3 G T G T C A T T C T T G A G T T T G A G T T T A G T C G C P04.5 C T C A A A C T C A A G A A T G A C A C A C G T A A C P26m-3 C G C C C C A G A A T C A T C T C T T T C C T C A T A T G G P26m-5 G A A A G A G A T G A T T C T G G G G C G A A G T A C A C G Pd l .3 A G C G T A C T T T C C T T C T C C G G G T C T T A C Pd l .5 G G A G A A G G A A A G T A C G C T T G G C C C C T G Pd2.3 A G C A T A C T T T A C G C G C C T G A A C T G T C T Pd2.5 A G G C G C G T A A A G T A T G C T T G G C C C C T G Pd3.3 A A G C G T A C T T C G G T C T T A C G C G C C T G A A Pd3.5 G T A A G A C C G A A G T A C G C T T G G C C C C T G Pd4.3 A G C G T A C T T T C T A T T C T C G A G T T T G A G Pd4.5 C T C G A G A A T A G A A A G T A C G C T T G G C C C C T G Od-3 T C C A T T G T T G A G T G T G A T T C G A G T A T C T G Od-5 A T C A C A C T C A A C A A T G G A A A G T C G G T T GO-3 C T T A C G T G T G T C A T T C T T G A G T G T G A T T C G A G T A G C GO-5 A A G A A T G A C A C A C G T A A G T T G G T T T G G C C C T T G G a The primers are shown in the 5' -to-3' orientation. 96 Table 3-4. Specific het-c primer sets, templates used for the 1 s t round of P C R to generate specific alleles. Al le le Template Primer sets p o l het-cOR R e d - p o l . 3 het-cPA P o l . 5 - s p l . 3 po2 het-c0R Red - po2.3 het-cPA P o 2 . 5 - s p l . 3 po3 het-cOR Red-po3.3 het-cPA Po3.5-spl.3 po4 het-cPA Red - po4.3 het-c0R P o 4 . 5 - s p l . 3 po5 het-c0R Red - po5.3 het-cPA P o 5 . 5 - s p l . 3 o d l het-cOR R e d - o d l . 3 het-cOR 0 d l . 5 - s p l . 3 g o l het-cGR R e d - g o l . 3 het-cGR G o l . 5 - s p l . 3 del3 het-cGR R e d - d e l 3 . 3 het-cGR D e l 3 . 5 - s p l . 3 p d l het-cPA R e d - p d l . 3 het-cPA P d l . 5 - s p l . 3 pd2 het-cPA R e d - p d 2 . 3 het-cPA P d 2 . 5 - s p l . 3 pd3 het-cPA R e d - p d 3 . 3 het-cPA P d 3 . 5 - s p l . 3 pd4 het-cPA R e d - p d 4 . 3 het-cPA P d 4 . 5 - s p l . 3 97 3.2.5 Generation of vector cassette for construction of chimeric alleles Plasmids carrying het-cOR and het-cPA clones were used for generating chimeric vector cassettes. Conserved restriction sites in het-c used for chimeric construction are shown in Figure 3-4. The het-cPA allele was cloned as aXba 1-EcoR I fragment into pCRII vector to give plasmid pCRPA (Saupe and Glass, 1997), which has two unique restriction sites, Stu I and Sal I, in the specificity domain. The plasmid was then digested completely with Stu I and Sal I. The large fragment of approximately 6 kb was purified from agarose gel and used as a het-cPA ( S t u l'Sal l) vector cassette that contains intact pCRII vector and het-cPA with the removal of the 246 bp Stu I - Sal I fragment that spans the specificity region. The het-cOR allele was cloned as aXba I-EcoR I fragment in the pCRII vector to give plasmidpCROR that has two unique restriction sites (EcoR V and Sal I) in the specificity domain. The plasmid was digested completely with EcoR V and Sal I and the large fragment of about 6 kb was purified from agarose gel. The purified fragment was used as a het-c0R ( E c o R v-sai i) v e c t o r c a s s e t t e that contains intact pCRII vector, het-cOR with the removal of the 801 bp EcoR V-Sal I fragment spanning the het-c specificity region. 98 HET-c variable region > > | S 3 3^ 3 •§ het-c?A Aer-c°* 1 1 L-L het-c GR Red Yellow Figure 3-4. Conserved restriction sites in het-c used for chimeric construction. The three conserved restriction sites, EcoR V , Stu I and Sal I were used for manipulating in-frame exchange of D N A fragments in the specificity region between het-cPA, het-cOR and het-cGR. The Xho I and Stu I sites in het-cPA and the Apa I site in het-cOR were used for identifying chimeric constructs. The positions of P C R primers are shown in this diagram. 99 3.2.6 Construction of chimeric alleles Chimeric constructs between het-c alleles were as described by Saupe and Glass (1997). The unique D N A fragment that contains the sequence encoding the 34 - 48 amino acid variable domain was exchanged in-frame between alleles. The principle is diagrammed in Figure 3-5 and applied to allelic chimeric constructions. Chimeric constructs were identified by restriction site differences within the exchanged fragment. The specificity region of het-cOR - type alleles was digested and confirmed to contain conserved Stu l-Sal I sites. Each of the Stu l-Sal I fragments was purified from agarose gel and subcloned into het-cPA(S,u U S a l l) vector cassette to make het-cPA{OR S l u l'Sal I } chimeric alleles. The specificity region of het-cPA - type alleles was digested and confirmed to contain conserved EcoR V and Sal I sites. Each of the EcoR V-Sal I fragments was purified from an agarose gel and subcloned into the het-cOR(EcoKV-Salv vector cassette to make het-c0R(PA EcoKV~ S a l l ) chimeric alleles. The Xho I restriction enzyme was used for identifying all the chimeric constructs. The specificity region of GR-type alleles was digested with either Stu l-Sal I or EcoR V-Sal I and subcloned into het-cPA(S'ul'Sall) and het-cOR(EcoRV-Sall) vector cassette. Restriction enzyme Stu I was used for identifying chimeric alleles het-cPA(GRStu l ' S a l l ) , and Apa I was used for identifying chimeric alleles het-cOR(GREcoRV-San). A l l the artificial alleles have unique Stu I and Sal I sites in the specificity domain, and thus the Stu l-Sal I fragments were cloned into het-cPA(Slu l ' S a l ! ) vector. A n Xho I restriction site located within the Stu I - Sal I fragment was used for identifying chimeric constructs. 100 101 Figure 3-5. Principles for chimeric construction and transformation assay for het-c specificity. The specificity region of naturally occurring and artificially constructed het-c alleles was exchanged with the region of wi ld type het-c allele. The chimeric constructs were transformed into het-c0R, het-cPA and het-cGR strains to assay specificity by monitoring transformant phenotype, growth rate and occurrence of hyphal compartmentation and death. 102 3.2.7 Cloning chimeric constructs into A', crassa transformation vectors Two types o f iV. crassa transformation vectors were used: pCB1004 with hygR as a selectable marker (Carroll et al., 1994) andpOKE103 wi thpan-2 + as a selectable marker (gift o f R. L . Metzenberg). Restriction maps and multiple cloning sites of both vectors are shown in Figures 3-6 and 3-7. Chimeric constructs in pCRII vector were digested with EcoR I and Xba I and then subcloned into bothpCB1004 andpOKE103 vectors. 3.2.8 Construction of strain CJ44 A het-c deletion strain X22-2 (het-60R het-cnul1 thr-2 a) was isolated from strain C9-2 (het-6OR het-cPA thr-2 a; Xiang, unpublished result). To obtain het-c null strain CJ44 (het-6PA het-c""l!; pan-2; arg-5 a), strain Xa-3 (het-6°R het-cPA; pan-2; org-5 A; Xiang unpublished) was used as a female and crossed with strain X22-2. Ascospores produced in the cross were collected from Petri dish lids, streaked on agar plate with the supplements pantothenic acid (PAN) and arginine ( A R G ) , and heat-shocked in a 65°C oven for 45 min. Plates were cooled to room temperature and then incubated overnight at 30°C. Germinated ascospores were picked from plates under a microscope and transferred to 1 m l o f Vogel ' s agar media (Vogel, 1964) plus P A N and A R G . Progeny were first screened for P A N and A R G requirements. The pan-2, arg-5 progeny were tested for mating type on a lawn offlA (FGSC4317) and fl a (FGSC4347) tester strains, followed by het-6 heterokaryon test with tester strains (Table 3-2). Six progeny that were het-6°R ; pan-2; arg-5 A were selected and used in het-c heterokaryon tests with het-c tester strains (Table 3-2). Strain CJ44 displayed dual compatibility with both het-cPA and het-cOR tester strains and thus was designated as a het-cnu" strain. 103 Figure 3-6. Plasmid map of pCB1004 (Carroll et al., 1994). 104 Figure 3-7. Plasmid map of pOKE103 (gift o f R. L . Metzenberg). 105 3.2.9 D N A transformation The specificity of the chimeric constructs was tested by transformation assays in N. crassa. For pCB1004 as a transformation vector, C9-2 (het-6°R het-cPA thr-2 a), C2-2-9 (het-6PA het-cOR thr-2 A) and FGSC 2193 (het-6°R het-cGR A) were used as recipient strains (Table 3-1). ForpOKE103 as a transformation vector, C15-la (het-6°R het-cOR; pan-2; arg-5; inl a) and Xa-3 (het-6°R het-cPA; pan-2; arg-5 A) were used as recipient strains (Table 3-1). Spheroplasts were prepared according to Schweizer et al. (1981) and stored at - 70°C until use. A modified procedure of the P. anserina transformation protocol was applied to N. crassa (S. J. Saupe, personal communication). Fifty m l o f spheroplasts were thawed on ice and then heat shocked at 48°C for 5 minute, followed by a 30 second incubation on ice. The spheroplasts were placed at room temperature for 10 minute before D N A was added. One pg of D N A was used for each 50 p i of spheroplasts. The mixture o f spheroplasts and D N A was incubated at room temperature for an additional 10 minute and then added to 1 m l of 40% P E G , 10 m M M O P S , 50 m M C a C l 2 . The mixture was further incubated at room temperature for 10 minute. Seven m l of pre-warmed top agar was added into the mixture and poured into bottom agar plates. Transformation plates were incubated at 30°C for three days. A population of fifteen colonies was transferred to individual plates to assay vegetative incompatibility by monitoring colony morphology, growth rate and occurrence of hyphal compartmentation and death. For co-transformation, 0.5 pg D N A each of pOKE103 construct and pCB1004 construct was used. Strain CJ44 (het-6PA het-cN; pan-2; arg-5 a) was used as a co-transformation recipient (Table 3-1). 106 3.2.10 Heterokaryon incompatibility tests Heterokaryon incompatibility tests were performed by spotting conidial suspensions of auxotrophic strains onto Vogel 's minimal agar medium (Vogel, 1964). Strains forming vigorous conidiating cultures after three days incubation at 30°C were considered compatible. Relative genotype and origin of the tester strains are listed in Table 3-2. 3.2.11 Growth rate measurements Linear growth rate of transformants was measured in race tubes as described by Jacobson et al. (1998). A colony cut from a transformation plate was placed at one end of a 40 m l glass tube containing 25 m l of medium. A l l tubes were incubated at 25°C. The starting point was marked as the leading edge of the colony after overnight growth; subsequent growth was recorded as the distance to the leading edge of the colony at - 24 hr intervals. Each experiment was performed in three replicates with different colonies from the same transformation experiment. 3. 2.12 Light microscopy The stain Evan's Blue (Direct Blue 53, CI23860, Aldr ich Chemical Co. , Milwaukee, Wisconsin) is excluded from cells with intact plasma membranes (Gaff and Okong'O-Ogola, 1971) and was thus used to identify dead and dying hyphal compartments. A transformant was inoculated onto a cellophane membrane layered on top of agar plates and incubated at 30°C. Cellophane membranes with mycelium adhering were removed from the medium, placed on glass slides and flooded with 1% Evans blue in water (w/v). After 5 min, mycelia were rinsed in distilled water, floated off the cellophane and onto a glass slide, and mounted with 90% glycerol in 0.1 M phosphate buffer (pH 7.2) under a cover slip. Samples were 107 examined under bright field illumination on a Olympus microscope and photographed Kodak T M A X - 4 0 0 film. 108 3.3 RESULTS A n exchange of an approximately 220 bp (amino acid position 223 - 294) Stu l-Sal I fragment in the variable domain between het-c alleles was shown to be sufficient to result in a switch o f allelic specificity (Saupe and Glass, 1997). This principle was used for chimeric construction in this study to investigate the het-c specificity of natural and artificial mutant het-c alleles. Chimeric constructs were introduced into het-cOR, het-cPA and het-cGR strains, and het-c specificity was monitored by colony morphology, linear growth rate and occurrence of hyphal compartmentation and death. 3. 3.1 Phenotypes of transformants with standard het-c alleles Typically, transformants with standard het-c alleles in N. crassa exhibit three classes o f phenotypes that are distinguished by the morphology o f transformants, linear growth rate and occurrence of hyphal compartmentation and death (Table 3-5). The colony morphology of the three classes of transformants is shown in Figure 3-8. Class I, compatible transformants (++), such as introduction of a het-c allele (e. g. het-cPA) into a recipient strain with the same het-c background (e. g. strain C9-2 het-cPA in Table 3-1), display vigorous growth and normal conidiation (Figure 3-8) that is indistinguishable from wild-type strains. However, the growth rate varies among different recipient strains. Compatible transformants in the het-cPA recipient strain have a growth rate of 6.0 cm / day; compatible transformants in either het-cGR or het-c0R recipient strains have a growth rate o f 4.9 - 5.2 cm /day (Figure 3-9). The growth rate differences between the class I transformants may be due to the background difference o f the recipient strains and are not 109 statistically significant (t test, a < 0.05). The only unexpected member o f the class I transformants was the het-cGR strain containing the het-cPA construct; the compatible phenotype in this case may be due to a modifier in the het-c strain (S. J. Saupe, personal communication; see discussion 3.4.1). The occurrence of hyphal compartmentation and death in these transformants was examined by Evan's blue staining. N o significant occurrence (< 1%) o f hyphal compartmentation and death was observed and the hyphae exhibit morphology of a wi ld type strain (Figure 3-10). Table 3-5. Three classes of phenotypes of het-c transformants Rating class Occurrence of death, hyphal compartments Linear growth rate (cm / day) Colony morphology Class I (++) N o hyphal death, wi ld type hyphal morphology >4.0 Conidiation. Vigorous growth. Class II (-) Budding-like hyphal morphology, hyphal death occurred after 2 days. 1.0-2.5 Little conidiation. Poor growth. Class III (--) Hyphal death occurred within 1 day. < 1.0 N o conidia (aconidial). Curly, severely inhibited hyphal growth. The class II transformants displayed an intermediate incompatible phenotype (-). The introduction of either het-cPA or het-cGR into a strain with het-c0R background (e.g., C2-2-9 in Table 3-1) or the introduction of het-cGR into a strain with het-cPA background (e.g., C9-2 in Table 3-1) gave this class of transformants. They showed a linear growth rate o f 1 - 2.5 cm per day (Figure 3-9), scarce conidiation (Figure 3-8), budding-like hyphal morphology (Figure 3-10) and hyphal compartmentation and death that occur after two days of growth. 110 The class III type (--) o f transformants show a severely incompatible phenotype characterized by slow-growth (linear rate < 1.0 cm / day; Figure 3-9), curly, flat aconidial morphology (Figure 3-8), and hyphal death occurring in >20% of the hyphal segments within one day o f growth (Figure 3-10). This class of transformants is represented by introduction of het-cOR into a recipient strain of either het-cPA or het-cGR background. Allele PA OR GR Recipient strains C2-2-9 orC15-l (OR) C9-2 or Xa-3 (PA) FGSC2193 (GR) Figure 3-8. Colony morphology of introduction wild-type het-c alleles (PA, het-c ; OR, het-c0R; G R , het-cGR) into a het-c0R {C2-2-9, C15-1), het-cPA (C9-2, Xa-3) and het-cGR (FGSC2193) background. i l l Recipient strains B into P A • into OR • into GR PA GR OR Alleles Figure 3-9. Average growth rate (cm/day) of standard (wild-type) het-c transformants. Experiments were performed in triplicates. Error bar indicates standard deviation. Geneotypes o f recipient strains: P A , het-cPA; OR, het-cOR; G R , het-cGR. Figure 3-10. Hyphal morphology and occurrence of hyphal compartmentation and death in the three classes of het-c transformants after 1 day and 3 days o f growth (Table 3-5). Hyphal compartmentation and death were visualized by staining with Evan's blue for 5 min (See Materials and Methods, section 3.2.11). Magnification 72X. 112 3.3.2 Amino acid sequence variability of het-c alleles To determine whether the amino acid sequences or the pattern o f insertion/deletion in the variable domain is most important for het-c allelic specificity, I took advantage of the amino acid sequence diversity in the variable domain o f naturally occurring het-c alleles (see Chapter 2, Figure 2-4). The specificity o f the natural alleles was assayed by the introduction of chimeric constructs into recipient strains of different het-c backgrounds. Twenty-four of the natural alleles (Figure 3-1), representing amino acid substitutions at multiple codon positions in the variable region and retaining the conserved Stu I, Sal I and EcoR V restriction sites, were selected for chimeric allele construction. The het-c alleles clustered into three het-c allelic types by their pattern o f insertions/deletions as previously described (see Chapter 2; and Saupe and Glass, 1997). Two regions in the het-c variable domain that show amino acid differences among isolates are denoted as region I and II (Figure 3-1). The 24 isolates fall into two groups based on amino acid variations within region I. The first group contains a M ( G ) E E R R G G ( Q ) H consensus sequence within region I. The second group o f alleles is much more variable in amino acid sequence in region I, but has a consensus of IH(Y)E(Q/K)K(N)ET(N/D)G(R/P/C)S(E/R) . Less amino acid variability is observed among the 24 isolates in region II, and in particular a F P H V G motif is completely conserved in this region among all alleles. Chimeric construction and specificity o f alleles in the three allelic groups are described in detail below. 113 3.3.3 Chimeric construction and specificity of het-c - type of alleles The amino acid sequence variability in the specificity region of the ten OR-type alleles is shown in Figure 3-1. Two types of consensus sequences are observed in the variable region I, I H E K E T G S and M ( G ) E E R R G Q H . Amino acids in region II vary at multiple sites. In order to distinguish chimeric constructs, the 220 bp of the Stu I - Sal I fragment of the specificity region o f all ten OR-type alleles was cloned into the het-cPA ( S , U I _ 5 f l / I ) vector cassette. Chimeric het-cPA ( 0 R S t u 1 ~ S a l l) constructs were identified by the absence of an Xho I restriction site, which is present in het-cPA and absent in the het-cOR specificity region (data not shown). Each chimeric construct was introduced into het-c0R, het-cPA and het-cGR strains (Table 3-1) and the phenotype of the transformants was examined compared to transformants carrying wild-type het-c alleles. Regardless of the amino acid sequence variability in region I (for example, alleles M3721 and Ndi6793; Figure 3-1) and region II, all the OR-type alleles showed the same het-c specificity. A l l the transformants displayed class I phenotype (compatibility) when each chimeric construct was introduced into a het-c0R strain. A l l gave class III (severe incompatibility) transformants when introduced into either het-cPA or het-cGR strains (Table 3-6). A l l the class I type of transformants had an average linear growth rates of 4.3 - 5.7 cm per day (Table 3-7), and displayed vigorous growth and normal conidiation phenotypes (representative phenotype is shown in Figure 3-11). Treatment o f hyphae in the class I transformants with Evan's blue did not show an occurrence o f a significant level of dead hyphal compartments (data not shown). 114 A l l the class III type o f transformants displayed severely incompatible phenotypes and had linear growth rates of < 0.5 cm per day (Table 3-7). These transformants showed a flat, curling, aconidial morphology (representative phenotype is shown in Figure 3-11). The occurrence of dead hyphal compartments could be detected within one day o f vegetative growth (data not shown). The number of dead hyphal compartments reached approximately 30% of the population after two days of growth. OR • • Table 3-6. Summary o f the phenotypes of transformants containing het-c - type chimeric constructs into N. crassa strains. Alleles Phenotypes of transformants in recipient strains of different het-c specificity het-cFA het-cUR het-cUK Gsp8241 — ++ — Gsp8242 — ++ . — Ncl824 — ++ — Nc847 ++ — Ns5940 ~ ++ — Ns963 — ++ — Ndi6793 — ++ — Sh2739 — ++ — Sbl903 — ++ ~ NH940 — ++ ~ Ni3721 ~ ++ — Nil 799 — ++ — Gsp8239 ++ ~ Classification of transformants was described in Table 3-5. 115 Table 3-7. Average rate o f growth (cm / day) (mean ± SD) of the transformants containing naturally occurring het-cOR - type chimeric constructs. A l l experiments were done in triplicates. Alleles Average growth rate of transformants in recipient strains of different het-c specificity het-c™ het-cUK het-c™ Gsp8241 0.2 ± 0.05 5.2 ± 0.40 0.2 ± 0.00 Gsp8242 0.2 ± 0.00 4.5 ± 0 . 3 5 0.2 ± 0.00 Ncl824 0.3 ± 0 . 1 0 5.5 ± 0 . 3 5 0.3 ± 0 . 1 2 Nc847 0.2 ± 0.05 5.2 ± 0 . 2 7 0.2 ± 0.03 Ns5940 0.2 ± 0.00 5.0 ± 0 . 2 3 0.2 ± 0.03 Ns963 0.4 ± 0 . 1 3 4.3 ± 0 . 3 2 0.3 ± 0 . 1 0 Ndi6793 0.3 ± 0 . 1 2 5.0 ± 0 . 2 6 0.2 ± 0.03 Sh2739 0.3 ± 0 . 1 3 5.3 ± 0.24 0.3 ± 0 . 1 0 Sbl903 0.3 ± 0 . 1 1 5.7 ± 0 . 5 5 0.3 ± 0 . 1 0 NH940 0.3 ± 0 . 1 0 4.8 ± 0.20 0.2 ± 0.00 Ni3721 0.2 ± 0.00 5.1 ± 0 . 1 2 0.3 ± 0 . 1 0 Nil 799 0.2 ± 0.00 4.6 ± 0 . 3 8 0.2 ± 0.00 Gsp8239 0.3 ± 0 . 1 2 4.8 ± 0.20 0.3 ± 0 . 1 0 Figure 3-11. Representative colony morphology of transformants containing het-cOR - type chimeric constructs, a, b, c, introduction of het-c0R chimeric constructs into recipient N. crassa strain het-c0R (C2-2-9), het-cGR (FGSC2193) and het-cPA (C9-2), respectively. 116 3.3.4 Chimeric construction and specificity of het-cGR-type alleles The specificity region of GR-type alleles lacks both the OR-specific and PA-specific insertions (Figure 3-1). Restriction enzyme digestion and sequence analysis (data not shown) indicated that the specificity regions of all the GR-type alleles lack both Apa I and Xho I sites that are present in the OR-specific and PA-specific insertions, respectively. These alleles all contain a similar motif in region I, although amino acid variability and a single amino acid insertion/deletion is observed in this region. In region II, amino acid variability occurs at one or two amino acid sites (i. e., T A / V T R / Q I T L ) in all GR-type alleles with the exception of Ndol692 ( A H T R K T L ) that has three to four amino acid residue differences from remaining of the GR-type alleles (Figure 3-1). The EcoR V-Sal I or Stu l-Sal I fragments o f the specificity region from the six G R -type alleles (Figure 3-1) were cloned into either the het-cOR(EcoRW ~ S a l l ) or het-cPA(Stul~ S a l l ) vector cassette. The het-c0R (GREcoKY-SalV chimeric constructs were confirmed by the absence of an Apa I site located within the OR-specific insertion region. The het-cPA ( G R St"I_ S a l r ) chimeric constructs were distinguished from het-cPA allele by the absence o f the Xho I site located within the PA-specific insertion region. Both het-cPA (GRS^-™V and het-cOR (^^Kw-Saii) c h i m e r i c constructs gave class I transformants when introduced into het-c spheroplasts and gave class II transformants when introduced into either het-c0R or het-cPA strains (Table 3-8). A l l the class I type transformants had vigorous growth with average growth rates of 4.0 - 4.8 cm per day (Table 3-9). N o significant (less than 1%) hyphal compartmentation or death was observed. A l l the class II transformants displayed intermediate incompatibility phenotype and had an average linear growth rates of 1.4 - 2.2 cm per day (Table 3-9) and scarce conidiation. Microscopic 117 examination of these transformants revealed abnormal budding-like hyphal morphology and the presence of dead hyphal compartments after approximately 48 hours o f growth (data not shown). Table 3-8. Transformation of chimeric constructs of naturally occurring mutant het-cGR - type alleles into 7Y. crassa. Classification of transformants was described in Table 3-5. Al le le Phenotype o f transformants in recipient strains o f different het-c specificity het-cM het-cUR het-cGK Ncl945 - - ++ Sh7140 - - ++ Ss2741 - - ++ Ncl455 - - ++ Ndol692 - - ++ Ndi6788 - - ++ 118 Table 3-9. Average growth rate (cm / day) (mean ± SD) of transformants containing het-c chimeric constructs. A l l experiments were done in triplicates. Al le le Average growth rate of transformants in recipient strains of different het-c specificity het-cFA het-cUK het-cUK Ncl945 1.6 ± 0 . 1 5 1.4 ± 0 . 1 0 4.6 ± 0 . 1 0 Sh7140 1.4 ± 0 . 1 0 2.2 ± 0.30 4.5 ± 0 . 1 2 Ss2741 1.7 ± 0 . 2 3 1.8 ± 0 . 2 0 4.7 ± 0 . 1 5 Ncl455 1.5 ± 0 . 1 5 2.0 ± 0.24 4.5 ± 0 . 1 2 Ndol692 1.8 ± 0 . 1 7 2.3 ± 0.30 4.0 ± 0 . 1 0 Ndi6788 1.7 ± 0.18 1.9 ± 0.18 4.8 ± 0.20 3.3.5 Chimeric construction and specificity of het-c - type alleles The PA-type alleles fall into either one o f two groups in region I (Figure 3-1) and also display a high degree of amino acid variability within region II. Differences in the specificity region of PA-type alleles as compared to the GR-type and OR-type o f alleles include (Figure 3-1): 1) they contain a large insertion; 2) they show a high degree o f variability in amino acid composition in the insertion region among the eight alleles; 3) an exceptional allele containing an insertion of a different length, Ndi5923, was identified. Due to the additional Stu I site in the PA-specific insertion region in all PA-type alleles as compared to OR-type or GR-type alleles, the EcoR V-Sal I fragment of the specificity region of the PA-type alleles was used to replace the same fragment from het-c0R for chimeric construction. The unique Xho I restriction site located within the PA-specific 119 insertion region was used to distinguish chimeric constructs of het-c0R(PA EcoRV~SalV from het-cOR alleles (data not shown). The specificity of naturally occurring PA-type alleles is summarized in Table 3-10. Wi th the one exception ofNdi5923 (named allele p26 later), all other het-cPA chimeric alleles gave class I transformants when introduced into the het-cPA and het-cGR strains, and gave class II transformants when introduced into het-cOR strains. The class I (compatible) transformants showed an average growth rate of 5.5 - 6.5 cm per day in the het-cPA recipient strain and o f 4.5 - 5.2 cm per day in the het-cGR recipient strain (Table 3-11). The growth rate differences of the class I transformants in different recipient strains are consistent with the observations of transformants containing standard het-c alleles (see section 3.3.1). The class II transformants showed an average growth rate of 1.4 - 1.9 cm per day (Table 3-11). Dead hyphal compartments were not observed in the class I transformants in either het-cPA or het-cGR recipient strains but occurred in the class II transformants after two days o f growth (data not shown). The allele Ndi5923 has a PA-specific pattern of insertion but is shorter (four to six codons shorter, depending on the reference allele). Unl ike the other PA-type of chimeric constructs, the Ndi5923 construct gave class II transformants when introduced into het-cPA, het-c0R and het-cGR strains. The class II transformants in the three recipient strains display a similar phenotype, characterized by linear growth rate at approximately 1.3 -1.8 cm per day (Table 3-11), abnormal budding-like hyphal morphology and the occurrence of dead hyphal compartments after approximately two days of growth (Figure 3-12). This observation indicates that allele Ndi5923 (p26) has a het-c specificity different from that of het-c , het-cPA or het-cGR - type alleles. 120 It is possible that the class II phenotype o f p26 transformants was due to self-incompatibility of the allele p26. To exam this possibility, chimeric constructs oip26 allele were introduced into a het-c null strain X22-2 (het-6°R het-cnu" thr-2 a). Only class I transformants were observed. These data indicated that the incompatible phenotype of the p26 chimeric transformants was not due to the self-incompatibility o f the p26 allele but due to a modification of het-c specificity. Heterokaryons between the X22-2 transformants containing the p26 allele with the het-c tester C8cl58 (het-6°R het-cPA pyr-4 A) and FGSC4571 {het-6°R het-cORpyr-4; ad3B Am54) (Table 3-2) displayed a class II incompatible phenotype (data not shown), which is consistent with the observation that p26 allele confers a novel het-c specificity. 121 Table 3-10. Transformation of chimeric constructs of naturally occurring het-c - type alleles into N. crassa. Alleles Phenotype o f transformants in recipient strains of different het-c specificity het-cFA het-cUK het-cUK NclBO ++ - ++ Nc4711 ++ - ++ Nc4832 ++ - ++ N12316 ++ - ++ Nt6583 ++ - ++ Ndi3228 ++ - ++ Ndi5923 - - -Np7221 ++ - ++ Ss2740 ++ - ++ Gsp8243 ++ - ++ Classification of transformants is described in Table 3-5. A l l the het-c specificity type alleles gave compatible transformants when introduced into het-c strain. It may be due to a modifier in the het-cGR strain (see discussion 3.4.1). Ndi5923 has a different insertion size in the PA-specific motif and demonstrated incompatibility with strains containing any of the three het-c allelic types. 122 Table 3-11. Average rate of growth (cm / day) (mean ± SD) of transformants containing het-cPA chimeric constructs. A l l experiments were done in triplicates. Alleles Average growth rate of transformants in recipient strains of different het-c specificity het-cPA het-cUR het-cUK Nci130 6.3 ± 0 . 1 5 1.5 ± 0 . 1 3 4.7 ± 0.15 Nc4832 5.5 ± 0 . 3 0 1.7 ± 0 . 3 2 4.8 ± 0.22 N12316 6.0 ± 0.22 1.9 ± 0 . 4 0 4.6 ± 0 . 1 5 Nt6583 6.2 ± 0.20 1.7 ± 0 . 2 7 4.7 ± 0.20 Ndi3228 5.9 ± 0 . 2 8 1.4 ± 0 . 1 0 4.5 ± 0.20 Ndi5923 1.8 ± 0 . 1 0 1.5 ± 0 . 4 0 1.7 ± 0 . 1 0 Np7221 6.2 ± 0.20 1.6 ± 0 . 2 3 5.0 ± 0 . 2 5 Ss2740 6.5 ± 0 . 3 5 1.4 ± 0 . 1 5 5.2 ± 0 . 3 5 Gsp8243 6.0 ± 0 . 1 0 1.4 ± 0 . 2 0 4.9 ± 0.27 123 Figure 3-12. Hyphal compartmentation and death of Ndi5923 (p26) chimeric construct introduced into different het-c strains after one day and three days o f growth. Dead hyphal compartments were observed by staining with Evan's blue for 5 min. a.,p26 construct introduced into het-c0R strain. b,p26 construct introduced into het-cGR strain. c,p26 construct introduced into het-cPA strain. Magnification 64X. 3.3.6 Summary of het-c specificity of naturally occurring alleles Chimeric construction and transformation assays indicated that all the OR-type and GR-type o f alleles confer the same specificity as identified in the standard het-cOR and het-cGR alleles (Ncl824 and Ncl945; Saupe and Glass, 1997), respectively. Wi th the exception of Ndi5923, all other PA-type alleles confer the same specificity as the standard het-cPA allele (Nell30; Saupe and Glass, 1997). These data indicate that the naturally occurring variability in region I and region II (Figure 3-1) does not affect het-c specificity. However, the allele Ndi5923, which has a PA-type insertion of a different size, confers a novel het-c specificity. These data suggest that a new het-c specificity could be gained by altering the size of the P A -type insertion or by altering the amino acid composition of the PA-specific insertion. 124 3.3.7 Artificial PA-type alleles with size variation in the PA-specific insertion A s described in the above section, allele p26 (Ndi5923) gave class II transformants when introduced into het-cPA, het-cOR and het-cGR recipient strains. It is possible that incompatibility is due to amino acid variation in the sequences in the PA-specific insertion motif. To examine this possibility, an artificial allele p26m derived from p26 by the addition of five amino acids, D S G A K , was constructed (Figure 3-13). The allele p26m conferred the same specificity as a het-cPA allele (Nell30). Thep26m chimeric construct gave class I (compatible) transformants when introduced into het-cPA and het-cGR strains and gave class II (intermediate incompatible) transformants when introduced into het-cOR spheroplasts (Figure 3-14). The class I transformants showed vigorous growth and a growth rate of 5.7 cm per day in het-cPA strain and 4.8 cm per day in het-cGR strain (Figure 3-15). The class II transformants showed inhibited growth and a growth rate o f 1.4 cm per day (Figure 3-15). PA V L I D M E E R R G G H - S PVFPHVGTATKLKLENRQFRRVRPGEGYDSGAKYAWPLVT P2 6 A L I D I H Q K E D R S K S R I F P H V G T R T K L K R E N G M F V P A P Y E E R - D YVWPLVT P2 6m A L I D I H Q K E D R S K S R I F P H V G T R T K L K R E N G M F V P A P Y E E R D D S G A K Y V W P L V T * * * * * * * * * * * * * * * * * * * * * * * * * Figure 3-13. Amino acid sequences comparison o f H E T - c ( N c l 130), P26, and P26m specificity region. Note that P26m has an addition of five amino acids, D S G A K . Asterisks indicate identical sites. 125 Allele P A p26m p26 Recipient strains FGSC2193 (GR) Figure 3-14. Phenotype comparison oi het-c , p26m, p26 transformants after three days of vegetative growth. Genotypes of recipient strains are shown in Table 3-1. P A , het-cPA; OR, het-c0R; G R , het-cGR. Recipient strains H into PA • into OR • into GR Figure 3-15. Average growth rate comparison of het-cPA, het-cOR and het-cGR transformants containing either het-c A,p26 or p26m chimeric constructs. Bar indicates standard deviation. A l l experiments were done in triplicates. Genotypes of recipient strains: P A , het-cPA (C9-2); OR, het-c0R (C2-2-9); G R , het-cGR (FGSC 2193). 126 The data from the naturally occurring het-c alleles suggested that the size of the insertion or the added amino acids affected het-c allele specificity. To investigate how the insertion size affects allele specificity, a set o f artificial het-c alleles, pdl,pd2,pd3 andpd4, were generated from het-cPA that contained different numbers of amino acids in the P A -specific insertion region (Figure 3-16). The pdl allele has a deletion of five amino acids (A, G , S, D , Y ) and has the same size of insertion in the specificity region as p26. The pd2 allele has a deletion of seven residues (A, G , S, D , Y , G , E) . The pd3 allele has a deletion of ten amino acids (A, G , S, D , Y , G , E , G , P, R) . The pd4 allele has the complete deletion of the fifteen amino acids in the PA-specific insertion motif. The composition of alleles pdl,pd2 andpd3 was confirmed by D N A sequencing (see Appendix 7.2). Allelepd4 was distinguished from het-cPA by the Stu I restriction site within the het-cPA - specific insertion region (data not shown). The phenotypes o f the transformants containing the four artificial het-cPA alleles are summarized in Table 3-12.1 describe the characteristics of vegetative growth of the transformants in the following paragraphs. PA VLIDMEERRGGH-S PVFPHVGTATKLKL •--ENRQFRRVRPGEGYDSGAKYAWPLVT GR VLIDIHEKETRSESRIFPHVGTATRITL-- - NNG --KLVWPLVT OR VLIDMEERRGGH- SPVFPHVGTDTRVTLRNDTRNNG- ----KSVWPLVT P26 ALIDIHQKEDRSKSRIFPHVGTRTKLKR • --ENGMFVPAPYEERD-- YVWPLVT PD1 VLIDMEERRGGH-SPVFPHVGTATKLKL - --ENRQFRRVRPGEG --KYAWPLVT PD2 VLIDMEERRGGH-SPVFPHVGTATKLKL ENRQFRRVRPG --KYAWPLVT PD3 VLIDMEERRGGH-SPVFPHVGTATKLKL--- ENRQFRRV --KYAWPLVT PD4 VLIDMEERRGGH-SPVFPHVGTATKLKL--- ENR --KYAWPLVT Figure 3-16. Amino acid sequence comparison o f H E T - c , H E T - c , H E T - c , P26, P D 1 , PD2 , PD3 and PD4 within the variable region. 127 When chimeric constructs were introduced into the het-c strain (FGSC2193),pdl, pd2, andpd3 gave class II transformants (Table 3-12). These class II transformants showed an average growth rate of 1.7 - 2.0 cm per day (Figure 3-17). The colonies displayed flat growth and little conidiation (Figure 3-18). Dead hyphal compartments occurred after 3 days of vegetative growth (data not shown). Introduction of allele pd4 that has a GR-type deletion pattern into het-cGR spheroplasts gave class I (compatible) transformants (Table 3-12). A l l four alleles gave class II transformants when introduced into a het-cPA strain (C9-2 or Xa-3). These transformants displayed the average growth rate o f 1.4 - 1.7 cm per day (Figure 3-17) with sparse conidiation (Figure 3-18). When hyphae from these transformants were stained with Evan's Blue, dead hyphal compartments were observed at three days of growth and hyphae showed an abnormal, budding-like morphology (Figure 3-19). Introduction ofpdl, pd2,pd3 andpd4 chimeric constructs into a het-cOR strain (C2-2-9 or C15-1) gave a variety o f phenotypes. The pdl,pd3 and pd4 alleles gave class III incompatible transformants (-- type), characterized by severe growth inhibition with an average growth rate of 0.5 - 0.7 cm per day (Figure 3-17), no conidiation, and the occurrence of dead hyphal compartments after 24 hr of growth (Figure 3-19). Unl ike standard het-c class III transformants (such as introduction of het-cOR into a het-cPA strain), colonies of these class III transformants did not display a typical curly-edged morphology (Figure 3-18). Thepd2 allele gave class II transformants (intermediate incompatibility), which displayed an average growth rate of 1.5 cm / day (Figure 3-17), little conidiation, and the occurrence of dead hyphae compartments at 3 days o f vegetative growth (Figure 3-19 and Figure 3-18). A s shown in Table 3-12, transformants containing allelespdl,pd2,pd3 exhibited different phenotypes from transformants containing standard het-cOR, het-cPA or het-cGR 128 alleles. Al le le pd4 showed specificity similar to het-c . However, introduction of allele pd4 into a het-cOR strain gave transformants that exhibited a class III phenotype (severe incompatibility); introduction of the het-cGR into a het-c0R strain gave transformants that exhibited only a class II phenotype (intermediate incompatibility) (Table 3-12). The severity of the incompatible reaction may be due to amino acid differences in the specificity domain betweenpd4 and het-cGR (Figure 3-16). Table 3-12. Summary of the phenotypes of the transformants o f het-c alleles p26,pdl, pd2, pd3 and pd4. Allele Phenotype o f transformants in recipient strains of different het-c specificity het-cPA het-cOR het-cGR het-cPA ++ ++ het-cOR ++ het-cGR _ _ ++ p26 _ _ pdl _ _ pd2 _ pd3 _ pd4 _ ++ ++, - and — represent the same phenotype as interpreted in Table 3-5. 129 P A pdl pd2 pd3 pd4 A l l e l e s G R O R Recipient strains H into P A • into O R • into G R Figure 3-17. Average growth rate comparison of pdl, pd2,pd3 andpd4 chimeric constructs transformed into P A (het-cPA), O R (het-cOR) and G R (het-c R) recipient strains. Bar indicates standard deviation. A l l the experiments were done in triplicates. P A , het-cPA. OR, het-c0R. G R , het-cGR. Recipient strains C9-2 or Xa-3 PA C2-2-9 or C15-! OR FGSC2193 GR Figure 3-18. Phenotypes of vegetative growth of transformants containing pdl,pd2,p pd4 constructs. Genotypes o f the recipient strains are shown in Table 3-1. OR, het-c . d3 and P A , het-cPA. G R , het-cGR. 130 Recipient strains Figure 3-19. Hyphal morphology and occurrence o f dead hyphal compartments when pdl, pd2, pd3 and pd4 constructs were introduced into het-cPA (C9-2 oxXa-3) and het-c0R (C2-2-9 or C15-1) strains. Hyphae were examined within 1 day of growth. Dead hyphal compartments were observed by staining with 1% Evan's blue for 5 min. Magnification 78X. 131 3.3.8 Generation of artificial alleles with both PA-specific and OR-specific insertions To further investigate how the pattern o f insertion affects het-c specificity, two artificial alleles pol and po2, with both OR-specific and PA-specific insertions, were constructed. Al le le pol was generated by the two sets o f primers red-pol.3 and pol.5-spl .3 using het-cOR and het-cPA as templates, respectively, for the first round o f P C R . The same strategy was used to generate allelepo2 except that primer sets were red-po2.3 and po2.5-spl.3 for the first round P C R . The pol and po2 alleles have the same insertion in the specificity region. The pol allele has the conserved N N G sequences from het-cOR allele while po2 has the E N R sequences from the het-cPA allele (Figure 3-20). The oligonucleotides pol .5 and po2.5 have a single nucleotide synonymous substitution to eliminate the Stu I restriction site in the PA-specific insertion region. This alteration allowed the identification of these two alleles in chimeric constructs. The D N A sequences of both alleles were confirmed (appendix 7.2). The introduction of both pol and po2 alleles into het-cPA, het-cOR, and het-cGR strains gave transformants displaying incompatible phenotypes (Table 3-14, Figure 3-21). Both het-cPA and het-cGR strains containing either pol or po2 constructs displayed class III incompatible phenotypes, described as severely inhibited growth (linear growth rate < 0.5 cm / day, Figure 3-22) and dead hyphal compartments after 24 hr o f growth (Figure 3-23). The het-c0R strain that contained either pol or po2 constructs showed class II incompatible phenotype. 132 P A V L I D M E E R R G G H - S P V F P H V G T A T K L K L E N R Q F R R V R P G E G Y D S G A K Y A W P L V T GR V L I D I H E K E T R S E S R I F P H V G T A T R I T L N N G K L V W P L V T OR V L I D M E E R R G G H - S P V F P H V G T D T R V T L R N D T R N N G K S V W P L V T P O l V L I D M E E R R G G H - S P V F P H V G T D T R V T L R N D T R N N G Q F R R V R P G E G Y D S G A K Y A W P L V T P 0 2 V L I D M E E R R G G H - S P V F P H V G T D T R V T L R N D T R E N R Q F R R V R P G E G Y D S G A K Y A W P L V T Figure 3-20. Amino acid sequence comparison o f P O l a n d P 0 2 with H E T - c P A , H E T - c 0 R and H E T - c G R . It is possible that the incompatible phenotype resulted from self-incompatibility of pol or po2. However, the introduction o f pol and po2 individually into a het-c null strain X22-2 (het-6°R het-cnuU thr-2 a) gave only wild-type transformants (data not shown). These wi ld type transformants displayed het-c mediated vegetative incompatibility in heterokaryon tests with tester strains CScr-29 (ro-7 un-24 het-6°R het-cPA pyr-4; inl a) and FGSC4571 (un-24 het-6°R het-c0R; ad-3A; nic-2; cyh-1 Am54) (Table 3-2) (data not shown). These data indicate that pol and po2 confer novel het-c specificities that are different from het-cPA, het-cOR, and het-cGR. To test whether pol and po2 confer identical or different het-c specificities, chimeric • R "T" constructs o f pol and po2 with different selectable markers (either hyg or pan-2 ) were co-transformed into a het-c null strain CJ44 (het-6°R het-c"u"; pan-2; arg-5 A). Only class I (compatible) transformants were obtained, displaying an average growth rate of 4.7 cm per day (see Table 3-15 in section 3.3.11). These data indicate that pol andpo2 confer the same het-c specificity, and that the specificity of pol and po2 is not affected by the amino acid residue differences in the N N K or E N R block (Figure 3-20). 133 Table 3-13. Summary of transformant phenotypes containing pol and po2 chimeric constructs. Al le le Phenotype o f transformants in recipient strain of different het-c specificity het-cUK het-cFA het-cUK het-cUK - - ++ het-cFA - ++ ++ het-cUK ++ ~ — pol - — ~ pol - ~ --Classification of-, ~ and ++ was described in Table 3-5. 134 PA OR Allele GR 1 1 I, H POl P02 Recipient strains C2-2-9 or C15-1 (OR) C9-2 or Xa-3 (PA) FGSC2193 (GR) Figure 3-21. Phenotypes o f transformants of mutant alleles pol and pc Genotypes of the recipient strains are shown in Table 3-1. OR, het-c R . P A , het-cPA -GR o2 chimeric constructs. G R , het-Recipient strains S into PA • into OR • into GR Figure 3-22. Average growth rate of transformants containing alleles pol and po2. Bar indicates standard deviation. Recipient strains: P A , het-cPA (C9-2 ox Xa-3); OR, het-c0R (C2-, R (FGSC2193). A l l experiments were done in triplicates. P A , het-2-9 or C15-1); G R , het-c cPA. OR, het-c0R. G R , het-cGR 135 Recipient strains Figure 3-23. Hyphal compartmentation and death in O R (het-c ) and P A (het-c ) transformants containing either pol and po2 constructs. The presence of dead hyphal compartments was assayed after 1 day of growth by staining with 1% Evan's blue for 5 min. Magnification 82X. 136 3.3.9 Alleles with chimeric combinations of amino acid sequence and patterns of insertion/deletion. Two polymorphic regions flanking the insertion/deletion motif are highly variable between het-c allele types (regions I and II in Figure 3-1). Genetic analysis (Saupe and Glass, 1997) and studies on naturally occurring het-c alleles indicated that the amino acid composition of polymorphic region I, MEERRGGH7IJTE(Q)KET(D)RS, does not affect het-c allelic specificity. To further investigate whether the amino acid composition of polymorphic region II, T D T R V T L / T A T K ( Q ) L ( I ) K ( T ) L , affects het-c allelic specificity, alleles with artificial combinations of polymorphic regions and patterns o f insertion/deletion were constructed (po3, po4 , odl and gol, Figure 3-24). One type, po3 andpo4, contains mosaic combinations between het-cPA and het-cOR. The allele po3 has a PA-specific insertion with an OR-specific polymorphic region II, T D T R V T L , while po4 has an OR-specific insertion with a PA-specific polymorphic region II, T A T K L K L . The second type of alleles (odl and gol) contains mosaic combinations between het-cGR and het-cOR. Al le le odl was generated from het-cOR with a deletion of the OR-specific insertion sequence, R N D T R ; while allele gol was generated from het-cGR with an addition of the OR-specific insertion sequence, R N D T R (Figure 3-24). The odl and gol chimeric constructs were identified by the unique Apa I site within the OR-specific insertion sequence. 137 PA VLIDMEERRGGH- SPVFPHVGTATKLKL E NRQFRRVRPGEG YDSGAKYAWPL VT GR VLIDIHEKETRSESRIFPHVGTATRITL NNG KLVWPLVT OR VLIDMEERRGGH- SPVFPHVGTDTRVTLRNDTRNNG KSVWPLVT P03 VLIDMEERRGGH-SPVFPHVGTDTRVTL ENRQFRRVRPGEGYDSGAKYAWPLVT P04 VLIDMEERRGGH- SPVFPHVGTATKLKL RNDTRNNG KSVWPLVT OD1 VLIDMEERRGGH - S PVFPHVGTDTRVTL NNG KSVWPLVT GOl VLIDIHEKETRSESRIFPHVGTATRITL RNDTRNNG KLVWPLVT Figure 3-24. Sequence comparison of the variable domain o f P 0 3 , P 0 4 , OD1 and G O l . B o l d region indicates polymorphic block II o f H E T - c 0 R and H E T - c P A . Transformation assays showed that po3 and po4 chimeric constructs have the same specificity as wi ld type het-cPA and het-c0R constructs, respectively (Table 3-14). Introduction of the po3 chimeric construct into the het-cOR strain gave class II type o f transformants that resemble the phenotype of a het-c0R strain containing a het-cPA allele (Table 3-14). Only class I transformants were obtained when the po3 chimeric construct was introduced into either het-cPA or het-cGR strains (data not shown). The introduction o f the po4 chimeric construct into het-cPA and het-cGR strains gave class III type of transformants. The phenotype of these transformants was similar to that of the introduction of the het-cOR construct into het-cPA and het-cGR strains. The introduction of either po4 chimeric constructs into het-c0R strain only gave class I transformants (data not shown). The introduction of odl and gol chimeric constructs gave transformants that had phenotypes resembling transformants containing het-c and het-c constructs, respectively (Table 3-14). The introduction of the odl chimeric construct into het-cOR and het-cPA strains both gave class II type of transformants (intermediate incompatibility) displaying an average growth rate of 1.4 cm per day; only class I (compatible) transformants were obtained when the odl chimeric construct was introduced into het-cGR strain. Introductions of gol chimeric constructs into het-cPA and het-cGR strains both gave class III type of transformants, displaying a severe incompatibility phenotype and an average growth rate of 0.3 cm per day; 138 while introduction of gol chimeric construct into het-c strain only gave class I (compatible) transformants displaying an average growth rate of 4.9 cm per day. Table 3-14. Summary of the phenotype of transformants containing po3, po4, odl and gol chimeric constructs. Classification of ++, - and — was described in Table 3-5. Al le le Phenotype o f transformants in recipient strains of different het-c specificity het-cOR het-cPA het-cGR het-cOR ++ het-cPA _ ++ ++ het-cGR _ ++ po3 ++ ++ po4 ++ odl ++ gol ++ 3.3.10 Summary of the het-c specificity of artificial alleles Artif icial chimeric het-c allele construction and transformation assays indicated that novel het-c specificities could be generated by altering the pattern o f insertion (i. e.,pol/po2) or the size o f the PA-specific insertion (i. e. pdl, pd2 and pd3) in the variable domain and that het-c allelic specificity was not affected by the exchange o f the polymorphic regions between alleles (i. e.po3,po4, odl and gol). Allelespol/po2,pdl,pd2 and pd3 displayed incompatibility with strains containing wild-type het-c alleles, het-cOR, het-cPA and het-cGR, 139 although the severity of incompatibility reactions differ. Whether pol/po2, pdl, pd2 and pd3 are mutually incompatible w i l l be described in section 3.3.12. 3.3.11 Deletion disrupting het-c activity A n allele del3 was generated by P C R from het-cGR that contained a deletion of the three amino acids N N G in the variable domain (Figure 3-25). The Stu I-Sal I fragment of the P C R product was cloned into het-cPA (StuI~Sal1) vector cassette and del3 was identified by the absence of an Xho I site (data not shown). The D N A sequence o f the del3 construct was verified by sequence analysis (see Appendix 7.2). Only class I transformants were obtained when the del3 chimeric construct was introduced into het-cOR, het-cPA or het-cGR recipient strains (Figure 3-26); hyphal compartmentation and death were not observed in any of the transformants (data not shown). These transformants had comparable growth rates with those of w i ld type het-c0R, het-cPA and het-cGR transformants, respectively (Figure 3-27). These data suggest that the three amino acids N N G are required for H E T - c function, protein folding or stability. A change o f the sequence N N G to E N R , however, does not affect het-c specificity as shown in the construction of alleles pol andpo2 (section 3.3.8). P A V L I D M E E R R G G H - S P V F P H V G T A T K L K L E N R Q F R R V R P G E G Y D S G A K Y A W P L V T OR V L I D M E E R R G G H - S P V F P H V G T D T R V T L R N D T R N N G K S V W P L V T GR V L I D I H E K E T R S E S R I F P H V G T A T R I T L NNG KLVWPLVT DEL3 V L I D I H E K E T R S E S R I F P H V G T A T R I T L KLVWPLVT Figure 3-25. Sequences comparison o f D E L 3 , H E T - c , H E T - c and H E T - c . D E L 3 was generated from H E T - c G R by deletion of N N G . 140 Figure 3-26. Phenotypes of transformants containing del3 construct. Genotypes of the recipient strains are shown in Table 3-1. OR, het-cOR. P A , het-cPA. G R , het-cGR. P A GR OR Al le les del Recipient strains B into PA • into OR • into GR Figure 3-27. Average growth rate (cm/day) o f transformants containing del3 construct. Bar indicates standard deviation. Recipient strains: P A (het-cPA), C9-2 ox Xa-3; O R (het-cUR), C2 2-9 or C15-1; G R (het-cGR), FGSC2193. Alleles: P A , het-c™; OR, het-cUK; G R , het-cUK; del, del3. A l l experiments were done in triplicates. PA. .OR. 141 3.3.12 Co-transformation of alleles with novel specificities The artificial alleles, pdl, pdl, pd3 and pol/pol (pol and pol confer the same specificity, see section 3.3.8), confer a different specificity from het-cPA, het-c0R and het-cGR alleles (Table 3-12 and 3-13). Do alleles pdl, pdl, pd3 and pol/pol confer the same specificity or display mutual incompatibility? To address this question, pair-wise combinations of these artificial alleles were co-transformed into a het-c null strain CJ44 (het-6°R het-cN; pan-2; arg-5 A) to assay incompatibility. W i l d type het-cPA, het-cOR or het-cGR constructs and the vectors pCB1004 and pOKE103 were used as controls. To determine i f the vectors used in het-c chimeric construction affected the phenotypes o f co-transformants, control experiments with vectors pCB1004 and pOKE103 were performed. Transformants containing vectors pCB1004 and pOKE103 (by co-selection ofhygR and P A N requirement) displayed class I phenotypes (Table 3-5), with vigorous growth, conidiating and an average growth rate o f 4.6 cm / day and no occurrence of dead hyphal compartments (Table 3-15). To examine i f the recipient strain CJ44 is an authentic het-c nul l strain, constructs of het-c alleles with defined specificity (i. e. het-cOR, het-cPA or het-cGR) were co-transformed into strain CJ44 by co-selection o f hygR and P A N requirement. Transformants containing constructs o f the same het-c alleles (i. e. het-cOR/het-cOR, or het-cPAlhet-cPA) displayed a class I phenotype (compatible; Table 3-15). Transformants containing constructs o f different het-c alleles, i . e. het-cPAlhet-cOR or het-cGRlhet-cOR, exhibited class III phenotypes (severely incompatible, Table 3-15), with curly, aconidial morphology, average growth rate of 0.3 cm per day and dead hyphal compartments within one day o f growth. These results were consistent with het-cOR/het-cPA and het-cOR/het-cGR partial duplication results (Saupe and 142 Glass, 1997). These experiments confirmed that the recipient strain CJ44 is a het-c null strain and that the co-transformation assay is working. To test i f alleles pdl,pd2,pd3 andpol/po2 are self-incompatible, two different constructs (in pCB1004 vector and in pOKE103 vector) o f the same allele were co-transformed into the CJ44 recipient strain to assay incompatibility. A l l o f the " s e l f co-transformants exhibited class I (compatible) phenotypes, with an average growth rate o f 4.3 -5.4 cm / day and no occurrence of dead hyphal compartments (Table 3-15). A s described in section 3.3.8, transformants containing constructs o f alleles pol and po2 showed class I phenotype (compatible phenotype), indicating that pol and po2 conferred the same het-c specificity. Transformants containing constructs o f pair-wise combinations of artificial het-c alleles, e. g., pol/pdl, pol/pd2, pol/pd3, pdl/pd2, pdl/pd3, orpd2/pd3, were co-selected for hygR and P A N requirements. A l l the transformants displayed a class III phenotype, showing severely inhibited growth (with an average growth rate of 0.3 - 0.6 cm per day), aconidial morphology and occurrence of dead hyphal compartments within one day of vegetative growth (Table 3-15). These results indicated that pol/po2, pdl, pdl and pd3 each confers a different het-c specificity. Thus, a number o f new het-c specificities were generated by altering the pattern of the insertion or the size o f the PA-specific insertion in the specificity domain. In the sequence comparison of all the het-c alleles, the artificial allele pdl shares the same pattern and size o f insertion in the specificity motif as the natural allele p26, although the amino acid compositions in the specificity region are variable (Figure 3-28). To examine i f pdl and p26 confer the same specificity, chimeric constructs o f pdl and p26 were co-143 transformed into the CJ44 strain. Only compatible transformants were obtained, suggesting that pdl and p26 confer the same het-c specificity. The polypeptides encoded by the alleles pd3 and het-c0R have the same size of the variable domain but have different amino acid sequences (Figure 3-29). The PD3 has five amino acid insertion, Q F R R K , in the PA-specific motif, while the H E T - c 0 R has a five amino acid insertion, R N D T R , in the OR-specific motif. Transformants containing both pd3 and het-cOR constructs displayed a class III (severe incompatibility) phenotype, which is consistent with the results observed when the pd3 construct was introduced into a het-cOR strain (Table 3-13). P2 6 ALIDIHQKEDRSKSRIFPHVGTRTKLKRENGMFVPAPYEERDYVWPLVT PD1 VLIDMEERRGGH- SPVFPHVGTATKLKLENRQFRRVRPGEGKYAWPLVT *** ** ****** **** ** * * * ***** Figure 3-28. Sequence comparison of P26 and P D 1 . Asterisks indicate identical sites. OR VLIDMEERRGGHSPVFPHVGTDTRVTLRNDTRNNGKSVWPLVT PD3 VLIDMEERRGGHSPVFPHVGTATKLKLENRQFRRVKYAWPLVT ********************* * * * * ***** Figure 3-29. Sequence comparison o f PD3 and OR. Asterisks indicate identical sites. 144 Table 3-15. Phenotypes o f co-transformants of pol/po2, pdl, pd2 and pd3 chimeric constructs into strain CJ44. Allele pairs incompatibility Growth Rale (cm / day) (mcan±SD) Hyphal compartmentation and death observed by Evan's blue staining PCB + POKE ++ 4.6 ± 0.20 Not significant - -„ , het-c011 - licl-c ' - -f+ 4.7 ± 0.20 Not significant het-cPA + het-cPA ++ 4.8 ±0.20 Not significant het-cUR + het-cPA Iwt-cm + hct-cc,H pdl + pdl ++ 0.3 ±0.10 0.4 ±0.10 4.3 ± 0.30 Significant within l.day of growth, more at 3 days of growth Significant within 1 day of growth, more at 3-days of growth"' -"• , Not significant pdl + pd2 ++ Not significant pd3 + pd3 '.•++'. Not'significant. pol +pol - , ++ 4.7 ± 0.57 Not significant po2 +po2 ' . .++ 4.8 ± 0.46 . Not significant pol '•-pol , • ++ 4.7 ±0.30 Not significant pol, + pdl pol. + pdl pol t pd3 pdl +pd2 pdl - pd3 pdl -r pd3 p!6 +pdl ++ 0.3 ±0.10 0.4 ± 0.06 r 0.4 ±0.10 0.5 ±0.12 0.4 + 0.05 0.6 ±0.10 4.5 ±0.35 • Significant within 1 day of growth, more at 3 days of growth Significant within 1 day of growth, more at 3 days of growth • - Significant within 1 day of growth, , more at 3 days of growth Significant within 1 day of growth, ' more.at 3 days of growth . ' Significant within 1 day of growth, * „ more at 3 days of growth Significant within 1 day of growth, more^ at 3 days of growth Not significant p26 +p2d 4.6 ±0.17 Not significant Pd3 + het-cm \t Significant within 1 day of growth, • more at;3 days of growth Not significant means hyphal compartmentation and death < 1% in a population. Significant hyphal compartmentation and death > 10% of a population. All the experiments were done in triplicates. Description of ++ and — phenotype is in Table 3-5. 145 3.4 DISCUSSION The difference in a variable region of 34 - 48 amino acids was shown to be sufficient to alter het-c specificity as previously shown by chimeric construction (Saupe and Glass, 1997). However, factors important for het-c specificity in the variable region were not determined. A peptide sequence comparison o f the variable region showed two polymorphic blocks (I and II in Figure 3-1), two independent insertion or deletion motifs (OR-specific five amino acid insertion and PA-specific 1 4 - 1 6 amino acid insertion) and variation in amino acid sequences among alleles (Figure 3-1). Genetic data from partial diploid analysis indicated that the polymorphic block did not contribute to het-c allele specificity (Saupe and Glass, 1997). In this study, I further investigated the determinants for het-c allele specificity via chimeric allele construction and in vitro mutagenesis. The data w i l l help to gain a general understanding o f how het-c allele specificity is determined and how recognition maybe mediated. 3.4.1 Variation in het-c mediated incompatibility phenotypes Genetic and mutational analysis suggested that vegetative incompatibility reactions resulted from the co-expression of different het-c alleles. Theoretically, most of the cells should have identical nuclei in any single transformant and thus should display a nearly identical incompatibility phenotype. Although hyphal growth rate and conidiation were affected throughout the colony, only approximately 30% of the hyphal segments displayed compartmentation and death. This phenomenon was also observed in incompatible het-c partial diploids (Jacobson et al., 1998). 146 Several possible interpretations could account for these observations. First, co-expression o f different het-c alleles may not be the only factor essential for inducing vegetative incompatibility reaction. Other modifier molecules may be required to facilitate the activity o f the het-c products. If the expression or/and subcellular compartmentation of these modifier molecules varies among hyphal segments, the degree o f the incompatibility reaction may also vary. The second scenario is related to the stability o f the het-c products, which could be affected by the physiology of the hyphal compartment. Third, a threshold level of the different het-c products could be required for triggering an incompatibility reaction, and this level may vary among hyphal segments. Whether the expression of het-c is regulated during different vegetative growth phases is unknown due to the difficulty o f detecting het-c expression by Northern analysis (this study and S. J. Saupe, personal communication). In the class II transformants (intermediate incompatibility), hyphal compartmentation and death do not occur until after two to three days o f vegetative growth, however, growth was significantly inhibited. This phenotype could be explained by a modifier in the recipient strains that may interact with the components involved in the incompatible reaction. For example, introduction of the het-cGR allele into a het-cPA strain gave class II (intermediate) incompatible transformants while introduction of the het-cPA allele into a het-cGR strain gave class I (compatible) transformants (this study and S. J. Saupe, personal communication). Since the het-cGR strain used in this study is not isogenic at other loci (L. Glass, personal communication), it is possible that differences in specific modifier in the het-cGR and het-cPA recipient strains may cause phenotypic differences in the transformants. 147 The effects of genetic background or modifier in the recipient strains on the phenotypes of transformants were also shown by comparisons o f individual transformants and co-transformants of alleles pol,pdl,pd2, or pd3 with het-cPA, het-cOR or het-cGR (data not shown). For example, co-transformants of het-cOR + pol, het-cOR + pd2, het-cGR + pd2, and het-cGR + pd3, displayed class III (severe incompatibility) phenotypes while individual transformants of either pol and pd2 into a het-c strain or pd2 and pd3 into a het-c strain gave only class II (intermediate incompatibility) phenotypes as shown in Table 3-12 and 3-13. One explanation for the display of different incompatible phenotypes could be due to the different genetic background between the two transformation systems, in particular the background interference (or modifications) of the recipient het-cOR and het-cGR strains. However, it is possible that the more severe incompatible phenotype in het-c co-transformants resulted from the high copy numbers and expression o f het-c constructs. 3.4.2 het-c allele specificity was not affected primarily by amino acid substitutions in the variable region I first addressed the question whether the amino acid sequence composition in the variable region was the critical determinant for het-c allele specificity using the 24 naturally occurring het-c alleles. Twenty-three of the 24 naturally occurring alleles fall into one of the three categories o f het-c allelic types as designated by the pattern of insertions or deletions first observed inN. crassa (Saupe and Glass, 1997). Within each allelic group, alleles differ in their amino acid sequence at multiple sites. A n exception to the 24 naturally occurring alleles, Ndi5923 (p26) has a different insertion size but the same insertion pattern as PA-type alleles. 148 Specificity o f the 24 naturally occurring alleles was assayed by chimeric allele construction and transformation in N. crassa strains of different het-c constitution. The results indicated that the alleles with an OR-specific insertion pattern (insertion of five amino acid residues R N D T R from position 54 to 59) or GR-specific deletion pattern (with neither PA-specific insertion or OR-specific insertion) conferred the same specificity as wi ld type het-cOR (Nc2489) or het-cGR (Nci945) allele, respectively. Thus, the amino acid diversity that occurs between OR-type and GR-type o f alleles does not alter het-c specificity. The PA-type alleles have the same overall insertion pattern (the numbers of amino acid residues vary from 14 -16) , but the amino acid sequences in the insertion motif vary among alleles. O f the seven PA-type alleles, three alleles (Np7221, Gsp8243 and Ss2740) have 16 amino acid residues in the insertion motif, two alleles (Nci 130 and Nt6583) have 15 amino acid residues in the insertion motif, and two (Nc4832 and Ni2316) have only 14 amino acid residues in the insertion motif. However, all seven alleles conferred the same specificity as the wi ld type het-cPA allele (Nci 130, Saupe and Glass, 1997). The Ndi5923 (p26) allele has only 11 amino acid residues in the PA-specific insertion motif. The p26 allele confers a het-c specificity that is different from the previously known three types o f het-c alleles, het-cOR, het-cPA and het-cGR. The novel het-c specificity of p26 could be due to either the different insertion size or the amino acid sequence variation or the combined effect of both in the insertion motif of P26. However, P26m, which has additional five amino acid residues to the specific insertion motif of P26, confers the same specificity as that of other het-cPA type o f alleles. These data suggest that the specificity of het-c is affected by the size of the insertion rather than by the amino acid composition in the PA-insertion motif. This conclusion is further supported by a comparison of the allele pairs, p26 and pdl, 149 that confer the same het-c specificity. Both P26 and PD1 have the same pattern and size of insertion, but the amino acid compositions in the insertion motifs are different. These results indicate that the novel specificity o f p26 resulted from the reduction in the insertion size rather than amino acid sequences in the insertion motif, supporting the observation that amino acid sequence composition is not the primary determinant of het-c allele specificity. However, since information is not available regarding how conserved amino acid residues in the variable domain affect H E T - c function, and because the specificity o f alleles with random compositions of the amino acid sequences in the variable domain was not tested, it is not possible to conclude that amino acid composition in the variable domain is not entirely irrelevant for H E T - c function. 3.4.3 The pattern and size of insertion or deletion in the variable region are the primary determinants for het-c allelic specificity Studies on the specificity of allele p26 suggested that it is possible to generate a set of novel het-c specificity types by reducing the size of the PA-specific insertion. In this study, novel het-c specificities were generated either by alterations in the pattern o f insertion (allele pol/po2) or by alterations in the size of the PA-insertion motif (alleles pdl,pd2 and pd3). Sequence comparison suggested that the new specificity could result from a particular pattern and size of the insertions/deletions in the variable domain or from alterations in the spacing (the numbers o f amino acid residues) in the variable domain. Interestingly, PD3 and H E T - c 0 R have the same number of amino acid residues in the variable domain (Figure 3-29) but allele pd3 confers a different specificity from het-c0R, suggesting that new specificities could be generated by amino acid substitutions in the variable domain. It is interesting to note that 150 alterations in the size of the PA-insertion motif do not always result in novel het-c specificity. For instance, Nc4832 is one amino acid shorter than N c i 130 and two amino acid residues shorter than Gsp8243 in the PA-specific insertion; however, alleles Nc4832, Nci 130 and Gsp8243 all confer the same specificity. In contrast, PD2 is two amino acid residues shorter than P D 1 ; alleles pdl and pd2 confer different specificities. H o w the specificities were generated and how the size of PA-specific insertion may affect het-c allelic specificity w i l l be discussed in Section 3.4.5. 3.4.4 Predicted protein structural characteristics of the hypervariable region The specificity domain of het-c is rather hydrophobic and contains short stretches of amino acids, such as sequence F P H V G that are conserved among all alleles. The secondary structures of H E T - c 0 R , H E T - c P A and H E T - c G R in this domain were predicted hypothetically by the Gibrat (Gibrat et al., 1987), Levin, (Levin et al., 1986), D P M (Deleage and Roux, 1987) and S O P M A (Geourjon and Deleage, 1994; 1995) prediction programs (http://www.ibcp.fr) and are shown in Figure 3-30. These programs have been shown to have accuracy rates o f approximately 68 - 70 % (Gilbrat et al., 1987; Lev in et al., 1986; Deleage and Roux, 1987; Geourjon and Deleage, 1994; 1995). A l l these programs are based on algorithms of prediction to assign secondary structures, using the homology between fragments of submitted sequence and sequences in the database o f protein conformations. Secondary structures of the H E T - c specificity domain predicted by the different methods agree with each other although the prediction methods use very different algorithms. In general, the predicted consensus structure of this domain for all three H E T - c peptides is characterized by a Hel ix - C o i l - Hel ix - C o i l - p-Sheet - C o i l - P-Sheet motif (Figure 3-30), 151 except that an extra helix structure is predicted in H E T - c near the PA-specific insertion. The highly conserved region upstream of the hypervariable domain (helix I and II in Figure 3-30) in all three H E T - c peptides is predicted to form a helical structure by all the methods. The conserved region downstream of the hypervariable domains (P-strand II) in all three H E T - c peptides is predicted to form a P-sheet structure. The two specific insertion motifs, PA-specific and OR-specific, are both predicted to form a coi l structure between two P-strands. In the case of H E T - c G R , the conserved four residues N N G K are predict to form a coil structure between two P-strands, which is similar to that o f the OR-specific or PA-specific insertion motif. 152 CD * U CO CJ CJ CJ o * o CJ CJ CJ CJ Ii. * Cd CJ Cd X Cd E-i Cd CJ CJ X CJ CD * Cd CJ H CJ CJ E-1 Cd CJ Cd CJ CJ > * Cd CJ Cd Cd Cd * Cd Cd Cd Cd Cd « 1 CM * Cd Cd CJ Cd Cd S * Cd CJ CJ Cd CJ * Cd CJ X CJ CJ Cd a CJ CJ CJ Cd a CJ CJ CJ * X CJ U CJ CJ CD * O E-i CJ CJ CJ CO U E-i CJ CJ CJ > Q 03 o E H CJ CJ ! H U CJ E H CJ CJ CD CJ CJ E H CJ CJ W CJ CJ CJ CJ CJ O * CJ CJ E H CJ CJ P< * U E-i E H CJ CJ « CJ CJ E H Cd CJ > * Cd CJ Cd Cd Cd Cd Cd X Cd Cd CU CD Pi Cd CO X Cd Cd b * X CJ X Cd X a X CJ X CJ CJ ( A « X CO X CJ CJ C O X X CJ X w X CJ X Cd X s * X CJ X Cd X X Cd X Cd X * X X X Cd X X X X X Cd X hel X X X X X hel < * X X X CJ X X X CJ CJ CJ O * CJ CJ Cd CJ CJ > * U CJ Cd CJ CJ Cd CJ CJ CJ CJ Cu * Cd o CJ CJ CJ CM * Cd CJ Cd Cd Cd > * Cd CJ Cd Cd Cd CM * O CJ CJ CJ CJ CO CJ CJ CJ CJ CJ X CJ E H CJ CJ CJ CD * CJ E H CJ CJ CJ CD * CJ CJ E H CJ CJ CM CJ CJ CJ CJ CJ CM X CJ X CJ CJ Cd X CO X X X Cd X CJ X X X 2 * X CJ X X X a Q X CJ X X X M * Cd Cd Cd X Cd X ya u * Cd Cd Cd X Cd > * Cd Cd Cd X Cd JS * Cd Cd Cd X Cd cd X CO X X X CJ * X CJ Cd X X >H CJ CJ Cd X CJ 5M CJ CJ E H CJ CJ CO CJ CJ E H CJ CJ CJ CJ E H u CJ * CJ E H X CJ CJ * CJ CJ CJ CJ CJ CM * CJ CJ X CJ CJ Q CJ CJ X CJ CJ Cd X X X CJ X * X CJ X X X E-i X X X X X X CJ X X X * X X X X X * X X X X X a X X X X X CD * X X CJ X X o> >4 * X X X X X CJ * X X X X X JS X X X X X l-H * X X X X X < * X X CJ X X Cd X X CJ X X 4 J < (fl C CM U • H I > 2 a (1) Cu CO CD a CD * 0 CJ CJ 0 CJ CD * CJ H u CJ CJ CM * Cd CO CJ CJ CJ E H Cd 0 CJ CJ CJ CD * Cd E H 0 H-1 0 * CJ CJ CJ CJ 0 E H Cd Cd Cd Cd Cd h—1 CD * CJ CO CJ CJ 0 > * Cd Cd Cd Cd Cd u CM * Cd CJ CJ Cd CJ * Cd Cd Cd Cd Cd u E H CJ CJ CJ Cd CJ CM * Cd Cd CJ Cd Cd JS W 1 CD * U Cd H Cd Cd * Cd Cd Cd Cd Cd E H Cd Cd Cd Cd Cd [> * Cd Cd Cd Cd Cd CQ > * Cd Cd Cd Cd Cd CO Cd Cd CJ CJ CJ * Cd Cd Cd Cd Cd « CJ CJ CJ CJ CJ CM * Cd Cd CJ Cd Cd * CJ CJ E H CJ CJ * Cd Cd Cd Cd Cd CJ E H E H CJ CJ [> * Cd Cd Cd Cd Cd CJ E H E H CJ CJ > hH * Cd Cd Cd Cd Cd Pi U CO E H O CJ Ui a CJ CJ CJ CJ H CJ CO E H CJ CJ 0 * O E H O CJ CJ Q CJ CJ E H CJ CJ 55 CJ E H CJ CJ CJ 55 CJ CJ H CJ CJ 55 a E H E H CJ CJ Pi Cd CJ X Cd Cd * Cd CJ O Cd CJ * Cd Cd Cd Cd Cd I D E H Cd Cd Cd Cd Cd E H Cd Cd Cd Cd Cd <D M * Cd Cd Cd Cd Cd H * Cd Cd Cd Cd Cd JS 1 CM Cd Cd Cd Cd Cd CM Cd Cd Cd Cd Cd E H Cd Cd Cd Cd Cd E H O Cd Cd CJ a < * Cd Cd X X X Q O CJ CJ CJ CJ E H Cd CJ CJ CJ CJ E H CJ CJ CJ CJ CJ O * CJ CJ CJ CJ CJ CD * CJ CJ H CJ CJ > * CJ CJ Cd CJ CJ > * CJ E H Cd CJ CJ X Cd CJ CJ CJ CJ X Cd CJ CJ CJ CJ CM * Cd CJ CJ CJ CJ CM * Cd CJ CJ CJ CJ CM * Cd CJ Cd CJ CJ CM * Cd CJ Cd CJ CJ M * Cd X Cd CJ Cd > * Cd CO Cd CJ CJ CM Cd O X CJ CJ CM * CJ CJ CJ CJ CJ CO X CJ CJ CJ O CO CJ CO CJ CJ CJ Cd X O X CJ CJ X CJ E H U CJ CJ CO CJ CJ E H CJ CJ CD * CJ E H O CJ CJ CM CJ CJ X CJ CJ O * CJ O E H CJ CJ E H CJ U X CJ CJ CM CJ CJ CJ CJ CJ Cd X CO X CJ CJ CM X CJ X O CJ « X CJ X CJ CJ Cd X CO X CJ CJ Cd X CJ X CJ CJ Cd X CJ X X X X CJ CJ X CJ CJ 2 * X CJ X X X H * X CJ X CJ CJ 0 X CJ X X X [—1 Q X CJ X Cd X M * Cd Cd Cd X Cd H * X Cd Cd Cd Cd hH * Cd Cd Cd Cd Cd * X Cd X Cd X > * Cd Cd Cd Cd Cd " u < * X Cd X Cd X J * Cd Cd Cd X Cd JS * X X X Cd X Cd X co X X X Cd X X X X X CJ * X CJ Cd X X CJ * X CJ X X X S H CJ u CJ X CJ > H X CJ CJ X CJ 2 O CJ E H U O 53 CJ CJ E H CJ O to CJ CJ E H CJ CJ CO CJ CJ E H CJ CJ O 0 O O CJ pH CJ CJ O CJ CJ * CJ E H X CJ CJ * CJ E H X CJ CJ CM * CJ CJ CJ CJ CJ CM * CJ CJ CJ X CJ CM * CJ CJ X CJ O CM * CJ CJ X X CJ p CJ CJ X CJ CJ Q CJ X X X X Cd X X X X X Cd X X X X X J * X CJ X X X ^ * X X X X X H X X X X X E H X X X X X X X CJ X X X X X CJ X X X J * X X X X X hH * X X X X X < * X X X X X < * X X X X X a X X X X X O X X X X X 0 * X X CJ X X CD * X X CJ X X hH * X X X X X u * X X X X X CJ * X X X X X JS CJ * X X X X X CM X X X X X CM X X X X X •H * X X X X X * X X X X X <: * X X u X X < * X X u X X Cd X X CJ X X Cd X X CJ X X CO CO CO 05 c 4-) c • U a) CM (0 CM (fl C CO 0 1^ • H CO O 1^ • H c 1 > 2 CM c 1 43 > 2 0 a • H a; CM O 0 • H CM CJ CQ CD a Q CO CJ CO CD Q a X X CO CO a <v CO a o CJ U I 'o O ffl X x u w ti .2 « >-H o '5b •a .a <4H o g a* <L> CO " 2 8? C J • f i M [ L S £ -S «> CO CO H ° el g . ^ . 8 H I .SP 3 fa H i > 153 3.4.5 Model for generation of het-c allele specificity Most of the residues in the helices and the P-strands have clustered hydrophobic side chains in all three H E T - c polypeptides (Figure 3-30, indicated by asterisks). Hydrophobic side chains are usually in the interior of protein molecules. It is possible that in the specificity region o f H E T - c , the two P-strands may pack tightly together in a antiparallel configuration through side chain hydrophobic interactions to form a stable framework o f protein conformation (Figure 3-31). The allele-specific variable domain (the coi l structure) may form a specific protruding loop (or a groove) to cover the surface of the compact structure. The two anti-parallel p-strands and the allele-specific loop (or groove) may thus form a specific conformation that mediates het-c allele specificity (Figure 3-31). The coi l loop (or groove) structure may vary between different het-c allelic types but is similar within each allelic group. The variable loop domain may be involved in protein-protein interactions (between H E T - c proteins or with other proteins) or may regulate the stability o f protein complex formation. Similar structures have been wel l demonstrated by crystalline studies with immunoglobulin molecules where the antigen interface consists o f hypervariable loops formed by two tightly antiparallel P-strands known as the complementarity determining region (CDR) (Davis et al., 1990; Creighton, 1993). Such a structure has also been shown in the T-cell receptor recognition surface of M H C class I molecules (Jones et al., 1998; Zhang etal., 1998). The H E T - c 0 R - type peptides are predicted to have the same secondary structure in the specificity region (data not shown), and thus the same conformations in the variable domain. A l l the H E T - c G R - type proteins are predicted to have the same conformations in the specificity domain as wel l (data not shown). In the case of H E T - c P A - types, all the 154 polypeptides are predicted to form similar secondary structure, although the amino acid sequences in the PA-specific insertion motif are highly variable (data not shown). It is possible that all the het-cPA - type proteins would have similar conformations in the variable domain as well . Therefore, explicitly, within each het-c allelic group, the proteins are predicted to have the same or similar conformations in the variable domain. The hypothesis also explains the generation of new het-c allele specificities. Alterations in the pattern of insertion (alleles pol and pol) or size of the PA-insertion motif (alleles pdl, pd2 and pd3) would result in the formation of a different loop domain (the V region/domain in Figure 3-30 and 3-31) between the two P-strands and thus new conformations in the variable domain that confer a new allele specificity. A deletion of the N N G from H E T - c G R ( D E L 3 ) results in the loss of the allele-specific coil region (underlined in Figure 3-30), which would explain the elimination of its allele specificity. A n artificial allele pd3 results in a polypeptide that has the same length o f amino acid residues in the variable region as H E T - c 0 R . However, allele pd3 confers a different specificity from het-c0R as shown by transformation assay (section 3.3.7). It could be explained by the differences in the predicted secondary structures o f PD3 and H E T - c 0 R (Figure 3-32). According to the predicted secondary structure, the conformation of PD3 in the variable domain would result in two shorter antiparallel p-strands and a short protruding coil structure o f four amino acid residues, which differs from that o f H E T - c O R . Differences in the size (by one or two amino acids) o f the PA-specific insertion in naturally occurring het-cPA - type alleles did not result in different het-c specificities. One could explain this by postulating that the transformation assay is not discriminating enough to differentiate these different het-c specificities. Alternatively, i f the variable loop domain is 155 involved in protein-protein interactions or regulating the stability o f protein complex formation, it could be explained by the strength of the interactions mediated via the variable loop domain. It is observed that the het-cPA - type alleles have either four or five hydrophobic residues in the variable loop (corresponding to the underlined region in Figure 3-30). If hydrophobic interaction is a force to stablize protein complex formation, one would predict that all H E T - c P A - type proteins would mediate similar dynamics in protein-protein interactions. Two hydrophobic sites, residues P / G and G , are conserved in all alleles (except Ndi5923). These two sites could be important for protein-protein hydrophobic interactions. This speculation could account for the different specificities of alleles pdl, pd2 and pd3. It is observed that PD2 has a deletion of the conserved residue, G , compared with P D 1 , and that PD3 has a deletion of the conserved hydrophobic residue, P, compared to PD2 . The model discussed here is hypothetical and is based on computer prediction and chimeric allele analysis. N o structural data are available to support these hypotheses of how het-c specificity is generated and that the variable motifs are involved in protein-protein interactions. However, these hypotheses can be further tested experimentally. 156 H E T - c P A H 3 V Figure 3-31. Proposed protein conformations in the variable domain for determining H E T - c specificity. The two anti-parallel P-strands (see Figure 3-30) may pack tightly together by hydrophobic interactions. The protruding loop structure on the surface is composed of the deletion/insertion region of H E T - c polypeptide (see Figure 3-30). See text for details. 157 CD * U CO C J C J C J o * C J C J C J C J C J fc * fc u H ac H E-i H u C J ac C J CD * H C J EH C J C J EH fc u H C J C J > * fc u H fc H * W fc H w H * fc H C J H H S * H C J C J H C J < •X W u X C J C J H C J C J C J C J H C J C J C J C J > * fc C J H fc H p i fc H X fc fc Pi W CO X H H b * X C J X W ac a X u X C J C J « X CO X C J C J X EH X C J ac H X C J X H ac * X C J X H ac X u X H ac * X X X W ac * X X X H ac EH X X X ac ac rt! * X X X C J ac E-i X X C J C J C J O * u C J H C J u > * C J C J fc C J C J SC fc C J C J C J a CM * H C J C J C J C J fc * w C J H H H > * w u H H H a. * u C J C J C J C J co u C J C J C J C J sc u H C J C J C J CD * C J C J C J C J CD * u C J EH C J C J C J C J C J C J C J a SC a X C J C J fc X CO X ac ac w X C J X ac ac 2 * X C J X ac ac P X C J X ac ac M * w fc U ac fc * fc fc H ac fc > * H H fc ac fc * fc H H ac H w X CO X ac ac u * X C J H ac ac >H C J u fc ac C J S3 u C J EH C J C J CO C J C J EH C J C J C J C J EH C J C J < * C J H ac C J C J CM * C J C J C J C J C J fa * C J C J X C J C J Q u C J X C J C J H X X X C J ac * X C J X ac ac H X X X ac ac X C J X ac ac * X X X ac ac < * X X X ac ac a X X X ac ac o * X X C J ac X * X X X ac X u * X X X ac X X X X ac X * X X X ac X < * X X u ac X w X X C J ac X 13 JS o CJ u CJ CJ CJ CD * CJ EH CJ CJ CJ fc * H co CJ CJ CJ EH H CJ CJ CJ CJ CD * fc EH EH CJ EH EH H U fc w w > * H H a a w * fc H a a H tu CM * H fc CJ w fc 1 J5 * W H w H H j> * H H u H H CO fc fc CJ CJ CJ « CJ CJ CJ CJ a CD * O CJ EH CJ a CJ B EH CJ a CJ EH EH CJ CJ > CJ CO EH CJ CJ H CJ CO EH CJ CJ Q CJ CJ EH CJ CJ CJ CJ EH CJ CJ tu w CJ BC H fc j * w fc fc fc w u EH w w H fc w i> H * H H H H fc 43 CA 1 Pi H H fc fc fc EH CJ H fc CJ CJ ca P CJ CJ CJ CJ CJ EH CJ CJ CJ CJ CJ CD * CJ CJ EH CJ CJ > * CJ EH H CJ CJ ac H a CJ CJ CJ CM * U CJ CJ CJ CJ fc * fc CJ fc CJ CJ > * fc CO H CJ CJ CM * CJ CJ CJ CJ CJ CO CJ CO CJ CJ CJ ac CJ EH CJ CJ CJ CD * CJ EH CJ CJ CJ CD * CJ CJ H CJ CJ Pi CJ CJ CJ CJ CJ Pi ac CJ ac CJ CJ u ac CO ac CJ CJ fc ac CJ ac SC SC 2 * ac CJ SC SC X p as CJ ac SC X l~] M w H w SC w i> * fc W w H a • S > * u H H H fc "u * fc fc fc SC fc J3 fc ac CO ac SC SC CJ * ac CJ H SC SC >H CJ CJ CJ SC CJ S3 CJ CJ EH CJ CJ CO CJ CJ EH CJ CJ ac CJ CJ CJ CJ CJ < * CJ EH ac CJ CJ CM * CJ CJ CJ CJ CJ fc * CJ CJ ac CJ CJ P CJ CJ ac CJ CJ H ac ac ac SC SC * ac CJ ac X SC EH ac ac ac X SC ac ac CJ SC X SC * ac ac X X SC rt! * ac ac X X SC a ac ac X X EC CD * ac ac u X SC X CD * ac ac SC X SC CJ * ac SC SC X SC J S Pi ac ac SC X SC * ac SC SC X SC < * ac ac CJ X SC u ac SC CJ X SC CQ CO a) a) CO CO n 4-> ti a p rd a rt! Pi a rt! a. u •rH CO o u •H CO 1 XI > 2 CM a I > 2 CM ti ft •H CD CM o o ft •H CM o 0 ca CD p co CJ CO CD p CO CJ o '5b <u o cm o CD Br <a cfl cu 3 .12 C CD g . 2 •2 ^ CO Q cu § 1 o i i o 1 cu a H U o o U a o l l U 5 . u 2 CQ cu i to M 158 3.4.6 The use of artificial het-c alleles to study protein interactions A number o f artificial chimeric het-c alleles were constructed in this study for identifying the critical determinants for allele specificity. Whether the hypervariable region is involved in protein-protein interactions was not addressed in this study. Biochemical approaches using co-immunoprecipitation and genetical approaches such as yeast two-hybrid assays are required. However, these artificial and chimeric het-c alleles constructed in this study w i l l be very useful for studying H E T - c interactions via yeast two-hybrid system and in structural studies. 159 Chapter 4 Heteromeric complex formation of HET-c proteins 4.1 I N T R O D U C T I O N Morphological features (Jacobson et al., 1998) and evidence o f nuclear D N A degradation (Marek et al., 1999) suggested that the cellular responses of het-c mediated vegetative incompatibility have features in common with programmed cell death (PCD) in animal and plant cells. However, the genetic and biochemical mechanisms mediating the vegetative incompatibility response remain largely unknown. Mechanisms mediating non-self recognition have been investigated in many biological systems, such as the mating systems in S. cerevisiae (Ho et al., 1994), S. commune (Specht et al., 1992) and U. maydis (Kamper et al., 1995), and the transcriptional activator Fos-Jun oncoproteins (O'Shea et al., 1992). Protein heterodimerization was commonly found as an important mechanism for mediating recognition in these systems. In P. anserina, it was also proposed that heteromeric complex formation of different het gene products is a mechanism triggering vegetative incompatibility response, although this hypothesis is based on genetic and mutational analyses (Begueret et al., 1994). In N. crassa, a strain carrying a mutated het-cOR allele displayed dual compatibility with both het-cOR and het-cPA strains; introduction of a mutated het-cOR allele into a het-cPA strain only gave compatible transformants (Saupe et al., 1996b). These studies indicate that het-c mediated vegetative incompatibility reaction requires the co-expression of different het-c alleles. Different mechanisms could be proposed on how het-c mediates non-self 160 recognition during vegetative incompatibility. One hypothesis is that non-self recognition during vegetative incompatibility is mediated via H E T - c heteromeric complex formation. Under this hypothesis, formation of a H E T - c 0 R and H E T - c P A heteromeric complex is associated with the incompatible phenotype of strains containing het-c0R and het-cPA alleles, regardless o f whether homomeric complexes between H E T - c protein themselves are formed or not. Alternatively, one could hypothesize that non-self recognition during vegetative incompatibility is not mediated via heteromeric complex formation but is a consequence of sequential regulatory events. In this case, one het-c product (e. g., H E T - c O R ) may regulate the function or expression of a different het-c product (e. g., H E T - c P A ) or other cellular factors, which may result in a dominant negative effect on vegetative growth; however, vegetative incompatibility in this case is not associated with H E T - c O R and H E T - c P A heteromeric complex formation. To distinguish between these two possibilities, I performed immunoprecipitation assays to examine i f H E T - c 0 R and H E T - c P A form a heteromeric complex. Whether H E T - c 0 R forms a homomeric complex during vegetative growth was also examined. Because of the high similarity of putative het-c allele products (Saupe and Glass, 1997), two molecular probes were used in this study to detect different H E T - c proteins: the green fluorescent protein (GFP) and the nine amino acid hemagglutinin (HA) tag. The GFP gene was fused with het-c0R, and the HA gene was fused with both het-c0R and het-cPA. G F P and H A have been widely used as reporters in heterologous expression systems via gene fusion. The green fluorescent protein (GFP) is a spontaneously fluorescent 27 kDa protein (238 amino acids) originally isolated from the jellyfish Aequorea victoria. It has elicited much interest as a reporter in heterologous systems for in vivo visualization of protein 161 subcellular localization in a wide variety of cell types and organisms, such as E. coli (Cody et al., 1993), Caenorhabditis elegans (Chalfie et al., 1994), Drosophila melanogaster (Wang and Hazelrigg, 1994) and the filamentous fungus, A. nidulans (Sievers and Fischer, 1997). The amino acid sequence ( Y P Y D V P D Y A ) of hemagglutinin, a protein o f the influenza virus, has been successfully used to create epitope-tagged proteins for subcellular localization in D. melanogaster (Surdej and Jacobs-Lorena, 1994) and A nidulans (Sievers and Fisher, 1997). To address the question of whether vegetative incompatibility phenotype is associated with H E T - c heteromeric and/or homomeric complex formation, the fusion construct het-cOR::GFP was co-transformed with fusion constructs het-cOR::HA or het-c0R(PA)::HA into a het-c null strain. Commercially available anti-GFP and ant i-HA antibodies were used in immunoprecipitation assay and Western blot analyses to detect the presence of H E T - c O R : : G F P and H E T - c : : H A fusion proteins, respectively. 162 4.2 MATERIALS AND METHODS 4.2.1 Strains and media N. crassa strains C2-2-9, C9-2, C15-la,Xa-3 and CJ44 (see Chapter 3, Table 3-1) were used for this study. Genotypes and origins of these strains are described in Chapter 3 (Table 3-1). Vogel ' s (Vogel, 1964) synthetic media was used for cultures with required supplements. A l l cultures were maintained at 30°C. 4.2.2 Antibodies Ant i -GFP antibody was obtained from Boehringer Mannheim Co. (Cat. No . 1814 460). It is a mixture o f two mouse monoclonal antibodies (clone 7.1 and 13.1). The concentration of stock solution is 400 ug / ml . A 1:1000 dilution was optimized for Western blot analysis. A final concentration of 4 ug / m l was used for immunoprecipitation assays according to the manufacture's recommendations. Rabbit anti-HA polyclonal antibody Y - l 1 was supplied by Santa Cruz Biotechnology Inc. (Cat. No . sc-805). The concentration of stock solution is 200 ug / ml . A 1:600 dilution was optimized for Western blot analysis. A final concentration o f 5 pg / m l was used for immunoprecipitation assays according the manufacture's recommendations. Peroxidase conjugate anti-mouse IgG (whole molecule) was developed in rabbits and supplied by S I G M A (Lot. No . 018H4822, Cat. No . A9044). The working concentration for Western blot analysis was at a 1:8,000 dilution. Peroxidase conjugate anti-rabbit IgG (whole molecule) was developed in goat and also supplied by S I G M A Chemical Company (Cat. No . 163 A6154). The working concentration for Western blot analysis was optimized at a 1:5,000 dilution. 4.2.3 Construction of fusion genes Recombinant D N A techniques and plasmid preparation were described as in Chapter 3 (Section 3.2.2). For the purpose of co-transformation, two N. crassa transformation vectors that have different selective markers, pCB1004 and pOKE 103 (see Chapter 3 Materials and Methods), were used for fusion gene constructions. The fusion gene het-c0R::GFP was constructed in the vector pOKE103 by standard D N A manipulations (Sambrook et al., 1989). Fusion genes het-cOR::HA and het-c0R(PA)::HA were constructed by R. Todd in the vector pCB1004 as described below in section 4.2.3.2. A l l the fusion genes were designed to be expressed under the A. nidulans constitutive promoter, trpC. However, for the use of convenient restriction sites, all the fusion constructs contain the entire trpC and also part of the het-c promoter. 4.2.3.1 Fusion gene between het-c0R and GFP The GFP clone, SmGFP (soluble modified G F P , clone CD3-326), supplied by C L O N T E C H Laboratories Inc. (Palo Al to , California) was used in this study and GFP refers to SmGFP in the following text. Modifications of the SmGFP were described by Davis and Vierstra (1996). The het-cOR::GFP fusion plasmid, pOKEpCOR::GFP, was constructed through several steps (Figure 4-1). First, the promoter trpC was cloned into pOKE103 at the Acc I /Xba I sites to generate plasmidpOKEpC. Second, an 800bp BamH I / Sac I fragment containing the entire GFP open reading frame was cloned into the plasmid pOKEpC to give 164 pOKEpCGFP. Third, a 2.7 kb Sea I/BamR I fragment containing approximately 160 bp of the promoter region and the C-terminal truncated open reading frame o f het-cOR was cloned into pOKEpCGFP at the EcoR V / BamR I sites to give pOKEpCORr.GFP'. A t this step, GFP was fused with the C-terminal of het-c0R at the BamH I site but was not in-ff ame. To make the het-c0R::GFP fusion in-frame, the plasmid pOKEpCORr.GFP' was digested with BamR I to generate a linear D N A fragment containing the BamR I overhang, C T A G , at both ends. A n end-filling reaction with Klenow I D N A polymerase (Berger and Kimmel , 1987) at room temperature was performed for 1 hr to generate blunt ends o f the linearized D N A . The reaction mixture was heat inactivated at 65° C for 10 min and ligated overnight to give plasmid pOKEpCORr.GFP in which both het-cOR and GFP were in frame. The D N A sequence of the fusion construct was confirmed by D N A sequencing (Appendix 7.3). 165 5GGA T C G A T C C A A G G A G A T A T A A C A A T G Figure 4-1. Outline of the het-c r.GFP fusion construction. The fusion construct contains both trpC and het-c promoters. Arrow in the sequences indicates BamR I cutting site. The BamR I overhang, C T A G , was filled with Klenow D N A polymerase I to generate blunt end double stranded D N A . Re-ligation of the het-cOR::GFP resulted in an in-frame fusion between het-cOR and GFP. 1 6 6 4.2.3.2 Fusion genes between het-c and H A tag The fusion genes het-cOR::HA and het-cOR(PA)::HA were made by Dr. R. Todd for the purpose of localizing H E T - c (R. Todd, personal communication). A description of the construction o f the plasmids is as follow. The het-c0R was cloned into the Bluescript plasmid pKS at the Xba 1/ Sac I sites to generate the plasmidpKSCOR. The 27 nucleotide HA tag was inserted into het-cOR prior to the stop codon by P C R mutagenesis. The plasmid pKSCOR was used as the template for P C R reaction. Two overlapping oligonucleotide primers named CHA1 and CHA2 were used. The sequences of the primers are: C H A 1 , 5 ' C T C G T A G A A G T A T A A C C T C C G A T G C T C G T C C G 3 ' and C H A 2 , 5' T A T T C G A A T A C G C G T G A G C T T G G T G A A G T A A T G 3 ' . The 5' end of the CHA1 oligonucleotide contains part of the HA tag sequences; the 5' o f the CHA2 oligonucleotide contains the rest o f the HA tag sequences, the het-cOR stop codon, T G A , and the 3 ' end sequences o f het-cOR. The P C R reaction was performed according to the standard protocol as described in Chapter 3 (section 3.2.4) except that annealing was done at 50°C andpfu Taq polymerase ( B R L , Bethesda research laboratory) was used to generate blunt end P C R products. The P C R products were ligated by standard protocol (Sambrook et al., 1989). The generated plasmid was called pKSCOR::HA (Figure 4-2). The D N A sequence of the fusion constructpKSCOR.HA was confirmed by D N A sequencing (R. Todd, personal communication). To generate the het-c0R(PA)::HA construe?, the approximately 2.44 kb Sea I / Kpn I fragment of het-cOR::HA fusion gene that contains the het-c specificity region was replaced with the approximately 2.48 kb Sea I / Kpn I fragment from het-cPA to generate the plasmid het-c0R(PA)::HA. Since both EcoR V and Sea I generate blunt ends, the approximately 3.6 kb Sea 11 Sac I fragment of het-c0R::HA or het-c0R(PA) v.HA was subcloned intopCBpC at 167 EcoR V / Sac I sites to give plasmidpCBpCOR::HA orpCBpCOR(PA)::HA, respectively (Figure 4-3). 168 Figure 4-2. Generating het-c ::HA and het-c ' J::HA fusion genes (constructed by R. Todd). 169 Sail Cla\ ATG Figure 4-3. Cloning het-c ::HA fusion gene into N. crassa expression vectorpCB1004. The construct contains a trpC promoter and 126 bp of het-c promoter. Plasmid pCBpCOR(PA)::HA was constructed by the same principle (constructed by R. Todd). 170 4.2.4 Preparation of cell extracts Approximately five compatible transformants or 20 - 30 incompatible het-c transformants were transferred into Vogel 's selective liquid media containing hygromycin B and arginine and maintained at 30°C for three to four days. Mycel ia from the cultures were harvested on Whatman N o . l filter paper through vacuum filtration. The following protocol was adapted from Peleg et al. (1996), with modifications. Approximately 0.5 g of mycelia was suspended in 2 m l of extraction buffer containing 20 m M T r i s - H C l (pH 7.5), 50% (v/v) glycerol, 1 m M P M S F , 1 m M E D T A , 0.1 m M D T T and 0.1% Triton-100. Mycel ia were ground with a Pyrex tissue grinder at 4°C until the mixture was homogenized. The homogenate was added to an equal volume of ice-cold acid-washed glass beads and incubated on ice for 30 seconds, followed by one minute vortex at high speed. The mixture was incubated on ice for 5 minutes. Ce l l debris was removed by centrifugation at 1,000 g untill the supernatant was clear. The clear supernatant was the total soluble cell extract and was used for immunoprecipitation experiments. 4.2.5 Immunoprecipitation Immunoprecipitation was performed using the Protein A Immunoprecitation kit (Cat. No. 1 719 394, Boehringer Mannheim) following instructions in the manual with some optimization of the conditions. A l l incubation steps were performed at 4°C with gentle mixing. Five hundred p i of cell lysate was pre-absorbed with 3 p i o f calf serum for 60 minutes and then incubated with 40 p i of protein A-Sepharose for an additional 60 minutes. This eliminates non-specific immuno-binding to protein A - Sepharose in the lysates. The mixture was centrifuged at 2,000 rpm for 30 seconds and the supernatant was transferred into 171 a new Eppendorf tube. Five to twelve p i o f antibody (specifically, 12 p i of anti-HA polyclonal antibody Y - l 1 or 5 p i of pooled monoclonal anti-GFP antibody) were added to the supernatant and followed by overnight incubation at 4°C. Forty-five p i of protein A -Sepharose was then added to the mixture with a further incubation for 3 hours. The mixture was centrifuged at 2,000 rpm for 30 seconds and the supernatant was removed. The pellet was washed twice with low salt buffer B (0.2% NP-40 ,10 m M Tris, p H 7.5, 0.15 M N a C l , 2 m M E D T A ) , one time with high salt buffer C (0.2% NP-40, 10 m M Tris, p H 7.5, 0.5 M N a C l , 2 m M E D T A ) , and finally with 10 m M Tris p H 7.5. During all washing steps, the mixture was collected by centrifugation at 2, 000 rpm for 2 minutes. After the last wash, the residual washing buffer was removed carefully with a gauge needle. The pellet was resuspended in 40 p i of SDS sample buffer (62.5 m M T r i s - H C l , pH6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol and 0.25% bromophenol blue). Sepharose was pelleted from the suspension by centrifugation at 2,000 rpm for 1 minute and the supernatant was used for Western blot analysis. 4.2.6 SDS-Polyacrylamide G e l Electrophoresis ( S D S - P A G E ) Immunoprecipitated samples were heated at 95 °C for 3 minutes. Approximately 20 p i o f each sample was subjected to S D S - P A G E according to modified Laemmli 's discontinuous buffer system (Laemmli, 1970; Laemmli and Favre, 1973) using a Bio-Rad M i n i - G e l II apparatus (Bio-Rad). The electrophoresis conditions used were specified by the manufacturer. A 7.5%) SDS-Acrylamide separating gel that allows effective separation of proteins between 200 and 40 kDa was used to separate samples containing the H E T - c : G F P and H E T - c : H A 172 fusion proteins. Broad range pre-stained molecular mass markers ( B R L or N E B ) were used described by the supplier. 4.2.7 Western blotting Transfer of protein to nitrocellulose membranes from Laemmli S D S - P A G E was accomplished using the Towbin buffer system (Towbin et al., 1979) and either the Bio-Rad Trans-Blot™ or M i n i Trans-Blot™ electrophoretic transfer apparatus (Bio-Rad). Transfer was accomplished at 30 V overnight. Nitrocellulose membranes (Protein™, Schleicher & Schuell) were first hydrated in distilled water and then equilibrated in Towbin's transfer buffer for at least 15 minutes before preparing the gel/membrane transfer sandwich. The efficiency of transfer was monitored by Coomassie Blue staining of the gel. The membrane with transferred proteins was rinsed twice with T B S buffer (50 m M Tris, p H 7.5, 150 m M NaCl ) and then incubated with 3% B S A in T B S at room temperature with gentle agitating. After 1 hour, the membrane was transferred into primary antibody solution and further incubated for 1 hour. The membrane was washed three times in T B S T (TBS plus 0.1% Tween 20) and once in 1% B S A - T B S for 10 minutes each. The membrane was then transferred into secondary antibody solution for 30 minutes at room temperature with agitation. After the membrane was washed four times with T B S T for 15 minutes each, specific protein bands were visualized using a B M Chemiluminescence kit (Boehringer Mannheim) or E C L ™ kit ( R P N 2106, Amersham Pharmacia Biotech., U K ) and Kodak X -Omat film. 173 4.3 RESULTS 4.3.1 Construction of fusion genes The predicted polypeptides resulting from the fusion gene constructs are shown in Figure 4-4. Fusion constructs pCBpCOR::HA, pCBpCOR(PA)::HA (both made by R. Todd) and pOKEpCORr.GFP were made at independent experimental times for the purpose of localizing H E T - c . Mutational analysis showed that the truncation of the H E T - c glycine rich C-terminus did not affect het-c incompatibility by transformation assays (Saupe et al., 1996b, and R. Todd, personal communication). In the construction of het-cORr.GFP fusion genes, the BamR I site in the 3'-end of the het-cOR and 5'end of the GFP gene was used for generating recombinant molecules. The H E T - c 0 R : : G F P fusion protein contains 714 amino acids of H E T -c 0 R , 266 amino acids o f G F P , with an additional six amino acids in-between that are from the end-filling o f the BamR I overhang, C T A G , and partial D N A sequences of the GFP promoter (Figure 4-1). The fusion protein is predicted to have a molecular weight o f approximately 128 k D a (Figure 4-4A). The fusion protein H E T - c 0 R : : H A contains the entire 966 amino acids of H E T - c O R p l u s nine amino acid residues of the H A epitope and has a predicted molecular weight o f approximately 127 kDa (Figure 4-4B). The fusion protein H E T - c 0 R ( P A ) : : H A contains 730 amino acids of H E T - c P A at the N-terminal and 255 amino acids of H E T - c 0 R at the C - terminal plus nine amino acid residues of the H A epitope and has a predicted molecular weight o f approximately 128 k D a (Figure 4-4C). 174 A . H E T - c O R : G F P 982 aa 128 kDa 714 982 H E T - c O R GFP B. H E T - c O R : H A 975 aa 127 kDa 966 H E T - c O R H A tag C . HET-c O R ( p A >:HA 985 aa 128 kDa 730 985 H E T - c P A H E T - c O R H A tag Figure 4-4. Surnmary o f the predicted fusion gene products. 4.3.2 het-c activity of the fusion genes To confirm that the het-c fusion genes retain the capacity to mediate vegetative incompatibility and thus presumably would function like w i ld type het-c, fusion gene constructs were introduced into strains with different het-c specificities to assay incompatibility. The phenotypes of the transformants are summarized in Table 4-1. Introduction of fusion gene constructs pCBpCOR::HA or pOKEpCORr.GFP into the het-cPA strains C9-2 or Xa-3 (see Chapter 3, Table 3-1) gave class HI transformants that showed the same phenotype as introduction of het-c0R into a het-cPA strain. The transformants displayed severely inhibited growth, curly, aconidial colony morphology and occurrence of dead hyphal compartments. Introduction of the fusion gene construct pCBpCOR(PA)::HA into the het-c0R strain C2-2-9 (see Chapter 3, Table 3-1) gave class II (intermediate incompatibility) transformants that showed the same phenotype as introduction o f het-cPA into a het-cOR strain. The transformants displayed inhibited growth, abnormal hyphal morphology and occurrence of dead hyphal compartments after two days o f growth (data not shown). Only vigorous class I (compatible) transformants were obtained when fusion gene constructspCBpCOR.HA or pOKEpCORr.GFP were introduced into the het-c0R strains C2-2-9 or C15-la and when the fusion gene constructpCBpCOR(PA)::HA was introduced into a het-cPA strain C9-2. The class I transformants had the identical phenotype as introduction o f het-c0R into a het-cOR strain or introduction of het-cPA into a het-cPA strain, and displayed vigorous growth and no occurrence of dead hyphal compartments. These transformation results indicate that the het-c fusion gene constructs had retained wi ld type het-c activity and presumably mediated vegetative incompatibility in an identical manner to non-tagged het-c. 176 Table 4-1. Summary of the phenotypes o f transformants containing fusion gene constructs. Constructs Phenotype of transformants when the construct was introduced into different recipient strains C2-2-9 (het-c0R) C9-2 (het-cPA) C15-la (het-c0R, pan-2) Xa-3 (het-cPA, pan-2) pCB1004 ++ ++ ND ND pOKE103 ND ND ++ ++ het-cUR-pCB1004 ++ — ND ND het-c''A-pCB1004 - ++ ND ND het-cOR-pOKE103 ND ND ++ — pCBpCORr.HA ++ — ND ND PCBpCOR(PA)::HA - ++ ND ND pOKEpCORr.GFP ND ND ++ — Phenotypes of ++, - and — type of transformants were described in Chapter 3, Table 3-5. ND = not determined. 4.3.3 Heteromeric complex formation of HET-c proteins To examine i f H E T - c proteins form stable heteromeric complexes during vegetative incompatibility and/or stable homomeric complexes during vegetative growth, the fusion gene construct pOKEpCORr.GFP was co-transformed with pCBpCOR(PA)::HA or pCBpCORr.HA into the het-c deletion strain CJ44 (het-cN; pan-2; arg-5) by co-selection of hygR and P A N requirements. For the negative control experiments, het-cOR (in vectorpOKE 103) was co-transformed with het-c0R, het-cPA or het-cOR(PA)::HA (all in vector pCB1004) (see Chapter 3, Material and Methods), and the transformants were co-selected for hygR and P A N requirements. A n equal amount of cell lysates of these transformants was analyzed by two independent immunoprecipitation assays. To gain sufficient cell lysates for analyses, 177 pooled transformants (five compatible transformants or 20 incompatible transformants) were used. First, pooled (mixed) anti-GFP antibody was added to the total cell lysate for binding to Protein A-Sepharose. To ensure the efficiency of immuno-binding and immuno-reaction, immunoprecipitation samples were analysed redundantly with anti-GFP antibody by Western blotting (Figure 4-5). Equal amounts of protein o f immunoprecipitated samples from five sets of co-transformants, het-cOR:GFP/het-cOR(PA)::HA, het-cOR::GFP/het-cOR::HA, het-cOR/het-cPA, het-cOR/het-cOR(PA)::HA, het-c0R/het-c0R, were analyzed (the staining gel is not shown). A highly abundant band of approximately 55 k D a that corresponds to the molecular weight of the heavy chain of IgG is present in all samples. The presence of the corresponding Ig G band (55 kDa) confirmed the binding efficiency of the antibody to Protein A . The nearly identical density o f the 55 kDa band in all the lanes indicates that a nearly identical quantity of anti-G F P antibody was bound to protein A in all immunoprecipitation samples. Three bands of approximately 126 kDa, 98 kDa and 90 k D a are observed only in the transformants that contain the het-cOR::GFP fusion construct (lanes 1 and 2 in Figure 4-5). The 126 kDa band corresponds to the molecular weight of the predicted H E T - c O R : : G F P fusion protein. The 98 k D a and the 90 k D a bands could be the degradation products of the H E T - c 0 R : : G F P fusion protein (see 4.4 Discussion). The bands of 92 k D a to 96 k D a are present non-specifically in all samples with comparable density (see comments in next paragraph). To determine i f H E T - c proteins form stable heteromeric or homomeric complexes during vegetative growth, equal amounts of samples from the anti-GFP antibody immunoprecipitation assays were analyzed with rabbit anti-HA polyclonal antibody ( Y - l 1) by Western blotting (the staining gel is not shown). A s shown in Figure 4-6, two bands of 178 approximately 127 k D a and 98 k D a are present specifically in the sample from transformants containing the het-c0R::GFP and het-cOR(PA)::HA constructs (lane 1 in Figure 4-6). The 127 kDa band corresponds to the molecular weight of the predicted H E T - c O R ( P A ) : : H A fusion protein. The 98 k D a band may be the N-terminal degradation product o f the H E T - c 0 R ( P A ) : H A fusion protein. The presence of the 98 k D a band w i l l be discussed in Section 4.4. Two bands corresponding to 92 - 96 kDa are also present non-specifically in all the samples with comparable amount in this analysis (labeled NS) . The presence o f these non-specific bands may be due to contamination of non-specific proteins (such as protein-A - like proteins) in the immunoprecipitation samples that bind non-specifically to IgG. These results suggested that H E T - c proteins (both the entire protein and the degradation product) are likely to form a heteromeric complex only in the incompatible transformants that contained het-cOR::GFP and het-cOR(PA): :HA fusion constructs and that no homomeric complex was detected in transformants containing het-c0R::GFP and het-c0R::HA fusion constructs. The observation of H E T - c heteromeric complex formation in incompatible transformants was confirmed independently by immunoprecipitation assays with rabbit anti-OR H A antibody ( Y - l 1). Samples from the same five sets of co-transformants, het-c r.GFP/het-cPA(OR)::HA, het-cOR::GFP/het-cOR::HA, het-cOR/het-cPA, het-cOR/het-cPA(OR)::HA, het-c0R/het-cOR, were used for this assay. A s described above, to ensure the efficiency of the immunoprecipitation reaction, equal amounts of proteins of all samples were first analyzed redundantly with anti-HA ( Y - l 1) antibody by Western blotting (Figure 4-7; the staining gel is not shown). Two bands of approximately 55 kDa and 79 k D a are present in all samples. The 55 kDa band present in all samples corresponds to the molecular weight of the IgG heavy chain. The nearly equal density of the 55 k D a bands in all samples indicating equal quantity 179 of anti-HA antibody was bound to the protein A in all immunoprecipitation reactions. The 79 k D a band present in all the samples with nearly identical density could be the aggregation product o f IgG heavy chains and light chains (55 k D a + 25 kDa, the staining gel is not shown). Two bands o f approximately 127 k D a and 98 k D a are observed specifically and nearly equal in transformants that contain het-c::HA fusion constructs (lanes 1, 2 and 4 in Figure 4-7). The 127 k D a band present in lanes 1, 2 and 4 in Figure 4-7 corresponds to the molecular weight o f the predicted H E T - c : : H A fusion proteins. The presence o f the 98 kDa band w i l l be discussed in section 4.4. The anti-HA antibody immunoprecipitation samples were further analyzed by Western blotting with mouse anti-GFP antibody. A s presented in Figure 4-8, two bands of approximately 127 k D a and 98 k D a were present specifically in samples from incompatible transformants that contained het-cOR::GFP and het-cOR(PA)::HA constructs (lane 1 in Figure 4-8). N o specific band was observed in the transformants that contain het-cOR::GFP and het-c0R::HA constructs (lane 2 in Figure 4-8). The 127 k D a band correspondings the predicted molecular weight of H E T - c 0 R : : G F P fusion protein. A s previously mentioned, the 98 kDa band is likely the N-terminal truncation product of the H E T - c O R : : G F P fusion protein. The presence of the 98 k D a band w i l l be discussed in section 4.4. These data are consistent with the observation in the immunoprecipitation assay with anti-GFP antibody that H E T - c proteins form a heteromeric complex during vegetative incompatibility. 180 Figure 4-5. Western blot analysis of mouse anti-GFP immunoprecipitation samples with mouse anti-GFP antibody. Lanes 1 - 5 represents transformants containing different het-c constructs. 1, het-cOR::GFPIhet-cOR(PA)::HA. 2, het-cOR::GFP/het-cOR::HA. 3, het-cOR/het-cPA. 4, het-cOR/het-cOR(PA)::HA. 5, het-c0R/het-c0R. Numbers on the right indicate the marker for molecular weight (kDa). Linear arrow indicates the band (approximately 126 kDa) corresponding to the predicted molecular weight of H E T - c 0 R : : G F P fusion protein. Block arrow indicates the 55 kDa IgG heavy chain. N S , nonspecific proteins binding non-specifically to IgG. The lower bands in lane 1 and lane 2 (about 98 kDa and 90 kDa) may be the degradation products of the H E T - c 0 R : : G F P fusion protein (see discussion). 181 NS —_ [_ * I B ? IMBF MW - 120 - 98 - 86 - 64 Figure 4-6. Western blot analysis of mouse anti-GFP immunoprecipitation samples with polyclonal anti-HA antibody ( Y - l 1). Lanes 1 - 5 represents transformants containing different het-c constructs. 1, het-c0R:;GFPIhet-c0R(PA)::HA. 2, het-c0R::GFP/het-c0R::HA. 3, het-cOR/het-cPA. 4, het-cOR/het-cOR(PA)::HA. 5, het-cOR/het-cOR. Numbers on the right indicate the marker for molecular weight (kDa). Linear arrow indicates the band (approximately 127 kDa) corresponding to the predicted molecular weight of H E T - c : : H A fusion protein. N S , nonspecific proteins binding non-specifically to IgG. The lower bands in lane 1 (about 98 kDa) may be the degradation products o f the H E T - c : : H A fusion protein (see discussion). 182 1 2 3 4 5 MW — 1 2 0 * * » — 98 — 86 NS-Figure 4-7. Western blot analysis o f rabbit anti-HA ( Y - l 1) immunoprecipitation samples with anti-HA antibody ( Y - l 1). Lanes 1 - 5 represents transformants containing different het-c constructs. 1, het-cOR::GFPIhet-cOR(PA)::HA. 2, het-cOR:GFP/het-cOR::HA. 3, het-c0R/het-cPA. 4, het-cOR/het-cOR(PA)::HA. 5, het-cOR/het-cOR. Numbers on the right indicate the marker for molecular weight (kDa). Linear arrow indicates the band (approximately 127 kDa) corresponding to the predicted molecular weight of H E T - c : H A fusion protein. Block arrow indicates the 55 kDa IgG heavy chain. N S , nonspecific bands (approximately 79 kDa) or possibly the aggregation product o f IgG heavy chain and light chain. The lower band in lane 1, 2 and 4 (about 98 kDa) may be the degradation products o f the H E T - c : : H A fusion protein (see discussion). 183 1 2 3 4 5 MW *m*m — 1 2 0 ~~ 98 — 86 — 64 Figure 4-8. Western blot analysis of rabbit anti-HA ( Y - l 1) immunoprecipitation samples with anti-GFP antibody. Lanes 1 - 5 represents transformants containing different het-c constructs. 1, het-cOR::GFPIhet-cOR(PA)::HA. 2, het-cOR::GFP/het-cOR::HA. 3, het-c0R/het-cPA. 4, het-cOR/het-cOR(PA)::HA. 5, het-c0R/het-c0R. Numbers on the right indicate the marker for molecular weight (kDa). Linear arrow indicates the band (approximately 127 kDa) corresponding to the predicted molecular weight of HET-c : : G F P fusion protein. The lower band in lane 1 (about 98 kDa) may be the degradation products of the H E T - c 0 R : : G F P fusion protein (see discussion). 184 4.4 DISCUSSION 4.4.1 Non-self recognition is mediated via protein heteromeric complex formation Two independent immunoprecipitation assays were performed in this study with consistent results. Both support the hypothesis that H E T - c proteins form a heteromeric complex during vegetative incompatibility and that a stable homomeric complex is not formed during vegetative growth. The first immunoprecipitation used anti-GFP antibody (Figure 4-5 and Figure 4-6) and the second immunoprecipitation used anti-HA antibody (Figure 4-7 and Figure 4-8). In the first redundant Western blot (Figure 4-5), a band of approximately 126 k D a that corresponds to the predicted molecular weight of the H E T - c O R : : G F P fusion protein and two bands o f approximately 98 kDa and 90 k D a were present only in transformants containing the het-cOR::GFP fusion construct (lanes land 2 in Figure 4-5). In the second redundant Western blot (Figure 4-7), a band of approximately 127 k D a that corresponds to the predicted molecular weight of H E T - c 0 R : : H A and H E T - c 0 R ( P A ) : : H A fusion proteins and a band of approximately 98 k D a were present only in transformants containing het-c ::HA and het-cOR(PA)::HA fusion constructs (lanes 1, 2 and 4 in Figure 4-7). These data suggest that the 126-127 k D a band is likely the specific band of H E T - c fusion proteins and that the 98 kDa and the 90 k D a bands are probably degradation products o f the fusion proteins. In both Western blots analyses with alternative antibodies (anti-HA antibody in the first assay and anti-GFP antibody in the second assay, Figure 4-6 and Figure 4-8), two specific bands of approximately 127 kDa and 98 k D a were present only in transformants containing both het-cOR::GFP and het-cOR(PA): :HA constructs (lane 1 in Figures 4-6 and 4-8). 185 These results indicate that H E T - c and H E T - c can form a heteromeric complex, and that a H E T - c 0 R and H E T - c O R homomeric complex is not detectable. These data support the hypothesis that H E T - c heteromeric complex formation is the mechanism mediating non-self recognition, although the dynamics of H E T - c heteromeric complex formation are not clear. In Western blots, putative H E T - c fusion protein degradation products were observed. A specific band of approximately 90 kDa occurred in samples containing het-cOR::GFP fusion constructs in Figure 4-5 (lanes 1 and 2). It could be inferred that the approximate 90 k D a band is probably the N-terminal degradation product of the H E T - c 0 R : : G F P fusion protein due to sample manipulation, since the corresponding band did not appear in other blots. A band of approximately 98 k D a was present in all the blots (both compatible and incompatible transformants) containing het-c0R::GFP and/or het-c::HA fusion constructs. Since it is consistently present in all blots and all transformants containing het-c fusion constructs, it is probable that the 98 k D a protein is the degradation product of the H E T - c fusion protein. Since both H A and G F P were fused with H E T - c at the C-terminal, it is probable that the N-termini of the fusion proteins was truncated. This truncation apparently occurs at the same site in the H E T - c 0 R : : G F P and H E T - c 0 R ( P A ) : : H A and is approximately 220-230 amino acids distant from the N-terminal. In this case, however, the 98 k D a protein would retain the specificity region of H E T - c (see Chapter 1, Figure 1-7). The 98 k D a band is present in the same pattern as the predicted H E T - c fusion proteins (shown as 126 and 128 kDa in the blots), which indicates that a heteromeric complex was also formed between the 98 k D a products (truncated H E T - c 0 R : : G F P and H E T - c 0 R ( P A ) : : H A ) . However, no differences were observed in the presence of the 98 k D a band between compatible and incompatible transformants, suggesting that the protein truncation is not specific to the vegetative 186 incompatibility reaction. Possible explanations for this observation could be: 1) a secondary cellular function o f the truncated H E T - c protein; and 2) posttranslational modifications of H E T - c polypeptides. The H E T - c peptide is predicted to have a glycine rich C-terminal domain that is similar to glycine rich domains found in a number of extracellular or cell envelope proteins (Saupe et al., 1996b). It is possible that H E T - c might be localized in the cell wal l or could have a secondary cellular function as a structural protein of the cell wal l components. However, microinjection experiments conducted by Wilson et al. (1961) indicated that het-c vegetative incompatibility in N. crassa was mediated by cytoplasmic elements. The capability of heterokaryon formation indicated that strains with different het-c genetic background are capable o f cell fusion although the fusion cells might not be viable. These observations suggest that H E T - c within the cell wal l may not be important in triggering vegetative incompatibility reaction. Due to technical difficulties, we were unable to localize H E T - c protein (R. Todd, personal communication and this study). However, we can not rule out the possibility of dual functions of H E T - c . First, H E T - c functions as a cell wal l component, in which case heteromeric complex formation mediates non-self recognition and thus triggers the formation o f a specific cell wal l structure, such as a septal plug, during vegetative incompatibility. Second, H E T - c functions as a cytoplasmic component, in which case heteromeric complex formation also mediates non-self recognition but triggers cellular responses, such as cell organelle and nuclear D N A degradation during vegetative incompatibility. The dual functions, cell wal l component and cytoplasmic component, may require different forms of H E T - c polypeptides: N-terminal truncated (98 kDa) and non-modified 187 (-127 kDa) forms. Since the N-terminal truncation would not affect the specificity region and the leucine zipper motif, both forms would presumably be able to form heteromeric complexes during vegetative incompatibility. Due to the technical aspects in this study, it is likely that the cell wal l components in the cell lysates may be mostly eliminated. Also due to the fact that cellular response of vegetative incompatibility does not require a high expression level of H E T - c (Saupe and W u , personal observations), whether truncated H E T - c is localized in the cell wal l or cytoplasm could not be distinguished by Western blot analysis in this study. Whether the 98 k D a product is due to the N-terminal truncation o f the fusion protein could be determined by N-terminal sequencing, and whether the truncated H E T - c is a cell wal l component could be determined by a combination o f H E T - c N-terminal deletion and localization studies. In summary, this study supports the hypothesis that non-self recognition during vegetative incompatibility is mediated via H E T - c heteromeric complex formation. These data also indicate that H E T - c 0 R does not form a homomeric complex in the soluble component of the cytoplasm. Due to the elimination o f the cell wal l components in the cell lysates in this study, it can not be excluded that H E T - c 0 R may form stable homomeric complex in the cell wal l as a structural component. 188 4.4.2 Possible mechanisms for heteromeric complex formation The predicted H E T - c peptides contain a leucine rich heptad repeat motif with hydrophobic residues at positions a and d (Figure 4-9; Saupe et al., 1996b; Saupe and Glass, 1997). This motif putatively forms amphipathic cc-helices that are found as a dimerization motif (termed leucine-zipper or coiled-coil structure) in several transcriptional regulators and fibrous structural proteins (O'shea et al., 1989b, 1991, 1992; Lehrer and Stafford, 1991). Mutational analysis has shown that an in-frame deletion o f the leucine rich haptad repeat in het-c abolished its ability to mediate vegetative incompatibility (Saupe et al., 1996b). It is possible that the leucine rich coiled-coil motif in H E T - c is essential in mediating H E T - c heterocomplex formation. The 34-48 amino acid variable region in H E T - c was found to be sufficient for conferring allelic specificity (Saupe and Glass, 1997). Introduction of alleles pdl and pd2 that have alterations in the size of the PA-insertion motif into a het-cOR strain resulted in class m and class II transformants, respectively, which may be due to different heteromeric complexes formed between P D l - H E T - c P A and P D 2 - H E T - c P A (Chapter 3). Immunoprecipitation data indicated that different H E T - c proteins formed a heterocomplex, while the same H E T - c proteins did not form a homocomplex. These studies suggest that the specificity region of H E T - c may also be important in mediating heterocomplex formation and non-self recognition. 189 426 458 H E T - c 0 R I V R S I N N M I E K I P G L E S L L E K I S E T L T A F I L G L defgabcdefgabcdefgabcdefgabcdefga 436 468 H E T - C P A I V R S I N N M I E K I P G L E S L L E K I S E T L T A F I L G L defgabcdefgabcdefgabcdefgabcdefga 422 454 H E T - C G R I V R S I N N M I E K I P G L E S L L E K I S E T L T A F V L G L defgabcdefgabcdefgabcdefgabcdefga Figure 4-9. The putative leucine rich haptad repeat region of H E T - c polypeptides. The residues at positions a and d o f the heptad repeats are hydrophobic (bold). This haptad repeat putatively forms amphipathic cc-helices (leucine zipper or coiled-coil structure) (Saupe et al., 1996b; Saupe and Glass, 1997). Leucine zipper coiled-coil motif mediating protein heterodimerization has been demonstrated in several systems. A well understood example of protein heterodimer formation is the Fos-Jun protein interactions in which preferential heterodimer formation by the Fos and Jun leucine zipper regions is largely a thermodynamic consequence of Fos homodimer instability (O'Shea et al., 1992). Fos and Jun, which are the protein products of the nuclear proto-oncogenes c-fos and c-jun, bind to D N A as dimers and modulate transcription o f a wide variety of genes in response to mitogenic stimuli (Ransone and Verma, 1990). The Jun protein forms a stable homodimer that can bind to D N A , while the Fos protein does not form a stable homodimer to bind D N A . However, the Fos-Jun proteins preferentially form a heterodimer over homodimer thermodynamically (O'Shea et al., 1992). 190 Both Fos and Jun contain a single leucine zipper region that is necessary and sufficient to mediate preferential heterodimer formation. The leucine zipper regions from Fos and Jun are known to fold as two-stranded, parallel coiled-coil helices (O'Shea et al., 1989; 1991) that prefer to interact with each other rather than with themselves (O'Shea et al., 1992). Eight amino acids from the Fos and Jun proteins were identified that were sufficient to mediate specificity and preferential heterodimer formation, while acidic residues in the Fos leucine zipper region were found to destablize the Fos homodimer formation through thermodynamic interactions (O'Shea et al., 1992). In S. cerevisiae, the homeodomain proteins al and cc2 form heterodimers to repress haploid-specific genes in diploid cells and allow post-fertilizational development, while oc2 homodimers repress a-specific genes, al does not form homodimers (Herskowitz, 1989; Ho et al., 1994). Ho et al. (1994) identified 3, 4 -hydrophobic heptad repeats within the N-terminus of the al and cx2 proteins and proposed that these motifs mediate dimerization by two leucine zipper-like coiled-coils. Crystallographic studies of the al/oc2 heterodimer have revealed that hydrophobic interactions and several hydrogen bonds between the coiled-coil interface of al and al proteins primarily promote al/a2 heterodimerization and discourage al/al homodimerization ( L i et al., 1995). Leucine zippers mediating heterodimer formation was also shown in the yeast transcriptional activator G C N 4 (O'Shea et al., 1989b; 1991). Destablization of a homodimer were also shown in the case o f the tropomyosin aP heterodimerization (Lehrer et al., 1989; Lehrer and Stafford, 1991). A specificity region functioning in heteromeric complex formation and mediating non-self recognition has been described in several fungal mating systems. In U. maydis, the b mating-type genes have variable and constant regions (Kronstad and Leong, 1990). Yeast 191 two-hybrid and immunoprecipitation studies indicated that the N-terminal variable regions of the b E and b W proteins determined the specificity and also functioned in bE-bW heterodimerization and that interactions between the b E and b W proteins from the same allele did not occur (Kamper et al., 1995). In C. cinereus, specificity determinants o f the A mating-type proteins HD1 and H D 2 were localized to the N-terminal 160-170 amino acids that were also important for H D 1 - H D 2 heterodimer formation (Banham et al., 1995). In S. commune, the specificity region of the Y 4 and Z5 proteins were found playing an important role in both Y - Z recognition and heteromeric complex formations, while interactions between Y 4 and Z4 (a self combination) did not occur (Wu et al., 1996; Yue et al., 1997). The data presented in this study and protein secondary structure prediction (Chapter 3) plus mutational analyses (Saupe et al., 1996b) suggested a possible mechanism for mediating H E T - c heterocomplex formation (Figure 4-10). The model hypothesizes that the leucine rich coiled-coil motif provides sufficient general contacts to form H E T - c protein complexes, perhaps via hydrophobic interactions. N o preference between a homomeric complex and a heteromeric complex formation occurs, since the coiled-coil motif is very similar among H E T - c 0 R , H E T - c P A and H E T - c G R . However, the stability o f the complex may be regulated by the conformations o f the specificity domain o f the H E T - c proteins. Different conformations in the specificity domain between alternative H E T - c proteins may promote the dimerization to form heterodimer; identical or similar conformations in the specificity domain between two H E T - c proteins may destablize the dimerization and thus prevent homodimer formation. The stability o f the complex may be due to thermodynamics that are primarily contributed by hydrophobic interactions and/or polar interactions between H E T - c proteins or interactions with other proteins. 192 The region of H E T - c that mediates protein-protein interactions has not been defined in this study. Structural and genetic studies (e. g. yeast two-hybrid assays) are required to examine whether the leucine rich coiled-coil motif and the specificity domain are directly involved in H E T - c heterocomplex formation. It is possible that a specific region is not directly involved in protein-protein interactions and that it only regulates the equilibrium of the protein complex formation. It is also possible that H E T - c proteins do not directly interact at all; instead, they may interact with a third protein (a docking protein) that is required to form a H E T - c heterocomplex. However, these possibilities can now be tested experimentally. 193 PA PA OR OR GR GR E r - . y M unstable E b L i 3 C unstable unstable PA OR PA GR OR GR 3—t 3—E 3—C 3...c stable stable stable Figure 4-10. Schematic model for stable H E T - c heterocomplex formation. The leucine zipper motif (indicated by L in open box) may provide general interactions for protein complex formation. The conformation o f the H E T - c specificity region (indicated by different filled blocks for allele-specific proteins) may regulate the stability o f the protein complex, possibly via hydrophobic thermodynamics. A different conformation may promote heterocomplex formation; the same conformation may discourage homocomplex formation. 194 Chapter 5 Concluding Remarks 5.1 Summary To understand the biological role of vegetative incompatibility in fungal populations and the molecular and biochemical mechanisms of het-c mediated vegetative incompatibility, three fundamental questions were addressed in this thesis. First, is the het-c locus subject to balancing selection for the role o f non-self recognition? Second, what is the primary determinant for het-c allelic specificity? Third, is the non-self recognition mediated by the mechanism of protein heteromeric complex formation? The het-c locus displayed trans-specific evolution and long persistence of polymorphisms within the Sordariaceae. These data indicated that the het-c locus is under balancing selection presumably to mediate non-self recognition during vegetative growth. A n increased number and frequency of non-synonymous nucleotide substitutions in the het-c polymorphic domain revealed positive selection favoring the diversity of H E T - c peptide for its role in mediating non-self recognition. This study supports the hypothesis that vegetative incompatibility is the bona fide non-self recognition system in filamentous ascomycetes in the Sordariaceae, and perhaps in other filamentous fungi as well . Comparable evidence for the persistence of ancient polymorphisms at the het-c locus, mammalian MHC loci and plant S loci reinforces the view that balancing selection is an evolutionary mechanism common to different non-self recognition systems. The isolation of the specificity region from a large number o f naturally occurring het-c alleles enabled me to investigate whether amino acid substitution in the specificity 195 domain affected het-c allelic specificity. Chimeric construction and transformation assays indicated that substitutions of amino acid residues in the variable region do not affect het-c allelic specificity. Construction of artificial het-c alleles and transformation assays revealed that the pattern o f insertion and the size o f the PA-specific insertion are the primary determinants for het-c allelic specificity. Based on the data presented in this study and protein secondary structure prediction, a H E T - c protein heterodimerization model was proposed for mediating recognition during vegetative incompatibility. The immunoprecipitation assay results indicated that H E T - c 0 R and H E T - c P A form a heteromeric complex during vegetative incompatibility while a H E T - c O R homomeric complex is not formed (at least not in the soluble component) during vegetative growth. The data suggested that non-self recognition is mediated via the mechanism of H E T - c heteromeric complex formation during vegetative incompatibility. 5. 2 General model of how het-c mediates vegetative incompatibility Vegetative incompatibility reactions appear to have conserved features in filamentous fungi. In N. crassa and P. anserina, the vegetative incompatibility response involves common stages such as hyphal compartmentation, vacuolization and death (Begueret et al., 1994; Jacobson et al., 1998; N . L . Glass, unpublished results), suggesting that common mechanisms mediate vegetative incompatibility. In P. anserina, a "poison heteromeric complex" model, in which heteromeric complexes between the products of incompatible genes are lethal to the cell, has been proposed for mediating vegetative 196 incompatibility based on genetic and mutational studies (Begueret et al., 1994). However, no direct evidence is available to support this hypothesis. In N. crassa, mutational and genetic analysis indicated that the vegetative incompatibility response requires the co-existence o f two incompatible het-c alleles (Saupe et al., 1996; Xiang, unpublished results). A strain that has a mutated het-c allele can not display vegetative incompatibility (Saupe et al., 1996b). Immunoprecipitation results presented in this study suggested that het-c mediates recognition during vegetative incompatibility via the formation of heteromeric complex o f different het-c products (Chapter 4). Chimeric het-c allele construction and secondary structure prediction suggested that the specificity domain of het-c may be involved in the mechanism of stable H E T - c heteromeric complex formation. The stability o f the heteromeric complex may be regulated by the specificity domains from two alternative H E T - c proteins, which may be associated with the observation of different phenotypes in incompatible transformants (Chapters 3 and 4). Observations o f the phenotype variations in vegetative incompatibility (Jacobson et al., 1998; chapter 3) indicated that incompatibility response mediated by het-c may be via different mechanisms. First, as proposed in P. anserina, the H E T - c heteromeric complex may directly "poison" the cell; it may have a direct negative affect on cell growth. A threshhold level of the heteromeric complex may be required to have a lethal effect on cells and thus trigger hyphal compartmentation and death (Figure 5-1). If formation of the heteromeric complex does not reach the threshhold level, hyphae may display different incompatibility phenotypes. Second, the active H E T - c heteromeric complex may induce sequential cellular events and thus trigger the incompatibility 197 response (Figure 5-1). The heteromeric complex may induce incompatibility reaction via interacting with a third molecule (X) or via regulating the expression or activity of the molecule (Y) that is essential in regulating cell growth (or death). Indirect evidence exists to support this model. First, morphological and biochemical studies indicated that het-c mediated vegetative incompatibility has common features with programmed cell death (PCD) in animal and plant cells (Jacobson et al., 1998; Marek et al., 1999). Secondly, the severity of incompatibility reaction was different in het-c reciprocal transformants either between het-cOR and het-cPA or between het-cGR and het-cPA (Chapter 3). The different incompatible phenotypes could be due to different levels of heteromeric complex formation, interactions o f the heteromeric complex with different modulators (X) in the recipient strains or heteromeric complex regulating the expression or activity of different targeting molecules (Y) (Figure 5-1). 198 i n c o m p a t i b i l i t y i n c o m p a t i b i l i t y negatively affect cell growth incompatibility Figure 5-1. General model for het-c mediated vegetative incompatibility. The model depicts the heteromeric complex formation o f the products o f incompatible het-c alleles. The H E T - c heteromeric complex may trigger vegetative incompatibility by a lethal affect on cell growth, interacting with the modulator X , or regulating the activity or expression of the Y that may induce vegetative incompatibility (see the text for details). OR, H E T -199 5.2 Future directions The biological significance o f vegetative incompatibility and the molecular basis oi het-c allelic specificity have been examined in this study. H E T - c heteromeric complex formation is shown to be a mechanism for mediating non-self recognition during vegetative incompatibility based on the data o f the immunoprecipitation assay. However, many questions remain to be further addressed. The het-c locus in the Sordariaceae displayed trans-specific polymorphism and is shown to be under balancing selection. Is het-c conserved and does the het-c locus also function as a het locus in species other than in the Sordariaceae? Chimeric construction examined the molecular basis for het-c allelic specificity determination. However, whether the specificity domain is directly involved in recognition mediated by heteromeric complex formation and how differences in the specificity domain affect hetermeric complex formation need to be investigated by yeast two-hybrid and structural studies. The chimeric alleles constructed in this study may be very useful in addressing these questions. Where H E T - c is localized in the cell during vegetative growth and whether H E T - c is localized in a different cellular compartment during vegetative incompatibility need to be investigated. Truncation o f H E T - c at the N-terminus and the association with its dual functions were proposed from the data presented in immunoprecipitation studies. These hypotheses could be examined by protein sequencing and localization studies. The H E T - c : H A and H E T - c : G F P fusion constructs may be useful for these studies. How vegetative incompatibility was triggered by H E T - c heteromeric complex also needs to be addressed through genetic approaches, such as mutational 200 analysis for identifying suppressers and yeast two-hybrid analysis for identifying unknown molecules that may interact with H E T - c . In phylogenetic analyses, at least seven het-c allelic lineages were resolved among 39 naturally occurring het-c alleles (Chapter 2). However, only four types o f allelic specificity, het-cOR - type, het-cPA - type, het-cGR - type and p26 (Ndi5923) were identified by the transformation assay (Chapter 3). Do the allelic lineages in phylogenetic analyses reflect functional het-c allelic types? If the specificity domain of H E T - c regulates the equilibrium of the H E T - c heteromeric complex formation, a heteromeric complex may be formed between H E T - c proteins that were detected as the same specificity type but which were assigned to different allelic lineages. The transformation assay in this study may not be sensitive enough to identify the phenotypes resulting from such a heteromeric complex formation. A more discriminating assay, such as the yeast two-hybrid system is needed. Morphological and biochemical evidence indicated that the vegetative incompatibility response had features in common with programmed cell death in animal and plant systems (Jacobson et al., 1998; Marek et al., 1999). The critical question that needs to be addressed towards understanding the cellullar and biochemical mechanisms of vegetative incompatibility is: does vegetative incompatibility resemble the biochemical pathway as programmed cell death described in higher organisms, such as in C. elegans or mammalian cells? If not, what are the participants in the pathway? Since N. crassa is a model organism for genetic studies, using genetic approaches to dissect the biochemical pathway o f vegetative incompatibility might be very favorable. 6. 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Appendices 7.1 DNA sequences alignment for phylogenetic analysis SS274 0 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT Sbl903 CAAGGCTGGA ACACTACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Sb714 0 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT SS2741 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Nc4481 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT Ncl824 CAACGCTGGG ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG CTGCATCCAT Ni3721 CAACGCTGGG ACACCACCAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG CTGCATCCAT Gsp8239 CAAGGCTGGA ACACTACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Gsp8241 CAAGGCTGGA ACACTACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NS963 CAAGGCTGGA GCACCACAAG TGCCGGCTAT ATTCGTTTCA GTTTGGAGCG TTGCATCCAT Sh2739 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GTTTGGAGCG TTGCATCCAT Nd3319 CAGGCCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG TTGCATCCAT Nd6793 CAAGGCTGGA ACACTACAAG TGCCGGCTAT ATTCCTTTCA GCTTGCAGCG TTGCATCCAT Gsp8242 CAGGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT NC24 89 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT NC4 709 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT Nc84 7 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT Ncl693 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT N i l 940 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT Nil799 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGGAGCG TTGCATCCAT NS5940 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GTTTGGAGCG TTGCATCCAT Np7221 CAAGGCTGGA ATACCACCAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Gsp8243 GGAGGCTGGA AAACCACCAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATTCAT Ni2316 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Ni3290 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NC4 832 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NC1130 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Ntl270 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NC4 711 CAAGGCTGGA ACACCACAAG TGCCGGGTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Nd3228 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Nt6583 CAAGGCTGGA ACACCACAAG TGCCGGGTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NS5941 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GTTTGGAGCG TTGCATCCAT NC4486 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT Nd5923 CAAGGATGGA ACACCACCAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG TTGCATCCAT NC14 55 GAAGGCTGGG ACACCACAAG TGCCGGCTAT ATCCGTTTCA GGTTGCAGCG TTGCATCCAT NC9S7 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG TTGCATCCAT NC1945 GAACGCTGGG ATACCACCAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG CTGCATCCAT Ndil692 CAAGGCTGGA ACACCACAAG TGCCGGCTAT ATTCGTTTCA GCTTGAAGCG TTGCATCCAT Nd6788 GAACGCTGGG ATACCACCAG TGCCGGCTAT ATTCGTTTCA GCTTGCAGCG CTGCATCCAT NC4499 CAAGGCTGGG ACACCACAAG TGCCGGCTAT ATCCGTTTCA GCTTGCAGCG GTTCATCCAT 552740 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG Sbl903 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CCTGTGCGAG Sb7140 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG 552741 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG NC4481 TTCGGTCGTC TGTATACCAG TGGCTCCCAT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG NC1824 TACGGCCGTT TGTATACCAG TGGCTCACAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG Ni3721 TACGGTCGTT TGTATACCAG TGGCTCACAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG Gsp8239 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAAACA AGGAGTCTGA CCTGTGCGAG Gsp8241 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CCTGTGCGAG NS963 TTCGGTCGTC TGTATACCAG TGGCTCTCAT GGCAAAGGCA AGGAGTCCGA CCTGTGCGAG Sh2739 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG Nd3319 TACGGTCGTC TGTATACCAG TGGCTCCACT GGCAGATGCA AGGAGTGTGA CCTGTGCGAG Nd6793 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CCTGTGCGAG Gsp8242 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAAGCA AGGAATCCGA CCTGTGCGAG NC2489 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG NC4 709 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG Nc847 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG NC1693 TTCGGTCGTC TGTATACCAG TGGCTCTCGT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG Nil940 TTCGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG Nil799 TTCGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCCGA CCTGTGCGAG NS5940 TTCGGTCGTC TGTATACCAG TGGCTCTCAT GGCAAAGGCA AGGAGTCCGA CCTGTGCGAG Np7221 TACGGTCGTC TGTATACCAG CGGCTCTCAT GGCAAAGGCA AGGAGTCTGA CCTGTGCGAG Gsp8243 TACGGTCGTC TGTATACCAG CGGCTCTCAC GGCAAAGGCA AGGAGTCTGA CCTGTGCGAG Ni2316 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTATGA CTTGTGCGAG Ni3290 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG NC4 832 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG NC1130 TACGGTCGTC TGTATACCAG TGGCTCTCAT GGCAGAGGCA AGGAGTCTGA CTTGTGCGAG Ntl270 NC4711 Nd3228 NtS583 NS5941 NC4486 Nd5923 NC1455 NC967 NC1945 Ndil692 Nd6788 NC4499 TACGGTCGTC TACGGTCGTC TACGGTCGTC TACGGTCGTC TTCGGTCGTC TACGGTCGTC TACGGTCGTT TACGGTCGTC TACGGTCGTC TACGGTCGTT TACGGTCGTC TACGGTCGTT TACGGTCGTC TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGTATACCAG TGGCTCTCAT TGGCTCTCAT TGGCTCTCAT TGGCTCTCAT TGGCTCTCAT TGGCTCTCAT TGGCTCACAT TGGCTCTCAT TGGCTCTCAT TGGCTCACAT TGGCTCTCAT TGGCTCACAT TGGCTCTCAT GGCAGAGGCA GGCAGAGGCA GGCAGAGGCA GGCAGAGGCA GGCAAAGGCA GGCAGAGGCA GGCAGAGGCA GGCAGAGGCA GGCAGAGGCA GGCAGAGGCA GGTAGAGGCA GGCAGAGGCA GGCAGAGGCA AGGAGTCTGA AGGAGTCTGA AGGAGTTTGA AGGAGTCTGA AGGAGTCCGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA AGGAGTCTGA CTTGTGCGAG CTTGTGCGAG CTTGTGCGAG CTTGTGCGAG CCTGTGCGAG CTTGTGCGAG CCTCTGCGAG CTTGTGCGAG CTTGTGCGAG CTTGTGCGAG TTTGTGCGAG CTTGTGCGAG CTTGTGCGAG SS2740 Sbl903 Sb7140 SS2741 NC4481 NC1824 Ni3721 Gsp8239 Gsp8241 NS963 Sh2739 Nd3319 Nd6793 Gsp8242 NC2489 NC4709 NC847 NC1693 Nil940 Nil799 NS5940 Np7221 Gsp8243 Ni2316 Ni3290 NC4832 NC1130 Ntl270 NC4711 Nd3228 Nt6583 NS5941 NC4486 Nd5923 NC1455 NC967 NC1945 Ndil692 Nd6788 NC4499 GCCTTGAGGT GCCTTGAGGT GCCTTAAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCTTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCTTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCCTTGAGGT GCTTGGGCCA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCCTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGCCA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GCTTGGGACA GGCCCTTCAT GGCCCTTCAC GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAC GGCCCTTCAC GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAC GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT GGCCCTTCAT ACCCTTGAGG ACCTTGGAGG ACTTTGGAGG ACTTTGGAGG ACCCTGGAGG ACCTTGGAGG ACCTTGGAGG ACCTTGGAGG ACCTTGGAGG ACCCTGGAGG ACCCTGGAGG ACTCTTGAGG ACCTTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTTGAGG ACTCTTGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCCTGGAGG ACCTTGGAGG ACTTTGGAGG ACTTTGGAGG ACTTTGGAGG ACCCTTGAGG ACTTTGGAGG ACTTTGGAGG ACTTCCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTCCCCGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTCCCTGC ACTTCCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTTCCTGC ACTTCCCTGC ACTTTCCTGC ACTTTCCTGC C CAT AG C AAC CCACAGCAAC CCACAGCAAC CCACAGCAAC CCACAGTAAC CCACAGCAAC CCACAGTAAC CCACAGCAAC CCACAGCAAC CCACAGTAAC CCACAGTAAC CCACAGCAAC CCACAGCAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCATAGCAAC CCACAGCAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGTAAC CCACAGCAAC CCACAGCAAC CCACAGCAAC CCACAGCAAC CCACAGCAAC CCACAGCAAC CCACAGCAAC SS2740 Sbl903 Sb7140 SS2741 NC4481 NC1824 Ni3721 Gsp8239 Gsp8241 Ns963 Sh2739 Nd3319 Nd6793 Gsp8242 NC2489 NC4709 NC847 NC1693 Nil940 Nil799 NS5940 TATTGCGAAC TACTGCGAAT TACTGCGAAC TACTGCGAAC TACTGCGAGT TACTGCGAAC TACTGCGAAC TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAC TACTGCGAAT TACTGCGAGT TACTGCGAGT TACTGCGAGT TACTGCGAGT TACTGCGAGT TACTGTGAAT TACTGCGAGT TACTGCGAAT TGGCCCTAAT TGGTCCTGAT TTGCCCTGAT TTGCCCTGAT TGGTCCTAAT TTGCCCTGAT TTGCCCTGAT TGGTCCTGAT TGGTCCTGAT TGGTCCTAAT TGGTCCTAAT TGGCCCTAAT TGGTCCTGAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTTCTAAT TGGTCCTAAT TGGTCCTAAT GGATATCCAT CGACATGGAA CTACATTCAT CGACATTCAT CGATATGGAA CGACATTCAT CGACATTCAT CGACATGGAA CGACATGGAA TGACATGGAA TGATATGGAA CGACATCTAT CGACATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA TGACATGGAA AAAAAAGAAA GAGAGGCGAG GAAAAGGAAA GAAAAGGAAA GAAAGGCGGG GAAAAGGAAA GAAAAGGAAA GAGAGGCGAG GAGAGGCGAG GAAAGGCGAG GAAAGGCGGG AAAAATGAAA GAGAGGCGAG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGAG CCGGCGAG-GCCAGCAC-CCCGCTCT-CCCGCTCT-GCGGGCAT-CCGGCTCT-CCGGCTCT-GCCAGCAC-GCCAGCAC-GCCAGCAT-GCGGGCAT-CCGGCCGC-GCCAGCAC-GCGGGCAT-GCGGGCAT-GCGGGCAT-GCGGGCAT-GCGGGCAT-GCGGGCAT-GCGGGCAT-GCCAGCAT--GAGAGTCAA AGTCCA -GAGAGTCGA -GAGAGTCGA AGTCCA -GAGAGTCGA -GAGAGTCGA AGTCCA AGTCCA AGCCCA -GAGAGTCAA -ACCAGTACA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGCCCA 222 Np7221 Gsp8243 Ni2316 Ni3290 NC4832 NC1130 Ntl270 NC4711 Nd3228 Nt6583 NS5941 NC4486 Nd5923 NC1455 Nc967 NC1945 Ndil692 Nd6788 NC4499 TATTGCGAAC TACTGCGAAC TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAT TACTGCGAAC TACTGCGAAC TACTGCGAAC TACTGCGAAC TACTGCGAAC TACTGCGAAC TACTGCGAAC TGGCCCTAAT TGGCCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TGGTCCTAAT TTGCCCTGAT TTGCCCTGAT TTGCCCTGAT TTGCCCTGAT TGGCCCTAAT TTGCCCTGAT TTGCCCTGAT GGATATCCAT CGACATCTAT CGATATGGAA CGATATGGAA TGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGATATGGAA CGACATTCAT CGACATTCAT CGACATTCAT CGACATTCAT CGACATTTAT CGACATTCAT CGACATTCAT AAAAAAGAAA AAAAATGAAA GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG GAAAGGCGGG CAAAAGGAAG GAAAAGGAAA GAAAAGGAAA GAAAAGGAAA GAAAATGAAA GAAAAGGAAA GAAAAGGAAA CCGGCGAG--CCGGCCGC--GCGGGCAT--GCGGGCAT--GCGGGCAT--GCGGGCAT--GCGGGCAT--GCGGGCAT--GCGGGCAT--GCCAGCAT--GCCAGCAT--GCGGGCAT--ACCGCTCT--CCCGCTCT--CCCGCTCT--CCCGCTCT--ACCCCCGCCG CCCGCTCTCG CCCGCTCT---GAGAGTCAA -GAGAGTCAA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA AGTCCA -AAGAGTCGA -GAGAGTCGA -GAGAGTCGA -GAGAGTCGA CGAGAGTCAA CGAGAGTCGA -GAGAGTCGA SS2 74 0 ATCTTCCCCC ACGTTGGCGC TCATACTCGA CTCACACTC- AAGAAT Sbl903 GTCTTCCCCC ACGTTGGCAC AAATACTCGA GTCACACTGA GAAATGACAC ACGTAACAAT Sb7140 ATCTTCCCCC ACGTTGGCAC AGCTACTCGA ATCACACTC- AATAAT SS2741 ATCTTCCCCC ACGTTGGCAC AGCTACTCGA ATCACACTC- -- AATAAT NC44 81 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT NC1824 ATCTTCCCCC ACGTTGGCAC AGCTACTCGA ATCACACTCA GGAATGACAC ACGTAACGAT Ni3721 ATCTTCCCCC ACGTTGGCAC AGCTACTCGA ATCACACTCA GGAATGACAC ACGTAACGAT Gsp8239 GTCTTCCCCC ACGTTGGCAC AAATACTCGA GTCACACTGA GAAATGACAC ACGTAACAAT Gsp8241 GTCTTCCCCC ACGTTGGCAC AAATACTCGA GTCACACTGA GAAATGACAG AGTAAACAAT NS963 GTCTTTCCCC ATGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT Sh2 739 GTCTTTCCCC ACGTTGGTAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT Nd3319 GTCTTCCCCC ATGGTGTCAT CGCTAATCGT GTCACAGTCA GGAGTGACAC ACGTAATAAT NdS793 GTCTTCCCCC ACGTTGGCAC AAATACTCGA GTCACACTGA GAAATGACAC ACGTAACAAT Gsp8242 GTCTTCCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGAAAC ACGTAACAAT NC24 89 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT NC4709 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT NC847 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT NC1693 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT Nil940 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCG AAAATGACAC ACGTAACAAT Nil799 GTCTTTCCCC ACGTTGGCAC AGATACTCGA ATCACACTCG AAAATGACAC ACGTAACAAT Ns5940 GTCTTTCCCC ATGTTGGCAC AGATACTCGA ATCACACTCA GGAATGACAC ACGTAACAAT Np7221 ATCTTCCCCC ACGTTGGCGC TCATACTCGA CTCACACTC- AAGAAT Gsp8243 ATCTTCCCCC ACGTTGGCGC TCATACTCGA CTCAGATAC- GAAAAT Ni2316 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC- GAGAAT Ni32 90 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAAGTC- GAGAAT NC4832 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC- GAGAAT NC1130 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC- GAGAAT Ntl270 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC- GAGAAT NC4711 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC- GAGAAT Nd3228 GTCTTTCCCC ACGTTGGCAC TGCGACTAAA CTCAAACTC GAGAAT Nt6583 GTCTTTCCCC ACGTTGGCAC TGAAACTAAA CTCAAACTC- GAGAAT NS5941 ATCTTTCCCC ACGTTGGCAC TGAGACTATA CTCAAACTC- GAGAAT NC44 86 GTCTTTCCCC ACGTTGGCAC TGCGACTAAT CACAAACTC- GAGAAT Nd5923 ATCTTCCCCC ACGTTGGCAC TCGTACTAAA CTCAAACGC- - GAGAAT NC1455 ATCTTCCCCC ACGTTGGCAC AGCTACTCAA ATCACACTC AATAAT NC967 ATCTTCCCCC ACGTTGGCAC AGCTACTCAA ATCACACTC- AATAAT NC1945 ATCTTCCCCC ACGTTGGCAC AGCTACTCGA ATCACACTC- AATAAT NdilS92 ATTTTCCCAC ATGTTGGCGC ACATACTCGG AAAACCCTC- AAGAAT Nd6788 ATCTTCCCCC ACGTTGGCAC AGTTACTCGA ATCACACTC- AATAAT NC4499 ATCTTCCCCC ACGTTGGCAC AGCTACTCAA ATCACACTA- ACTAAT 552740 GGAAAGTTCA CGCGGGTACA GGGTGGGGAA AGAGACGATT CTAGTCCTGA CAAGTACGTT Sbl903 GGA -AAGTCGGTT Sb714 0 GGA -AAGTTGGTT 552741 GGA -AAGTTGGTT NC4481 GGA -AAGTCGGTT NC1824 GGA -- -AAGTCGGTT Ni3721 GGA -AAGTTGGTT Gsp8239 GGA -AAGTCGGTT Gsp8241 GGA -AAGTCGGTT Ns963 GGA -AAGTCGGTT Sh2739 GAA -AAGTCGGTT Nd3319 GGA -AAGTCGGTT Nd6793 GGA -AAGTCGGTT Gsp8242 GGA-- -AAGTCGGTT NC24 89 GGA -AAGTCGGTT 223 NC4709 GGA -AAGTCGGTT NC847 GGA -AAGTCGGTT NC1S93 GGA - -AAGTCGGTT Nil940 GGA -AAGTTGGTT Nil799 GGA -AAGTTGGTT NS5940 GGA -AAGTCGGTT Np7221 GGAAAGTTCT CGCGGGTACA GGGTGGGGAA AGAGACGATT CTAGTCCTGA CAAGTACGTT Gsp8243 GGAAAGTACT CGCGGGTACA GGGTGGGGGA AGAGACTATT CTAGTCCTGG CAAGTACGTT Ni2316 AAACAGTTCT TGCCCACAAG GCCTGGAGAA CACGATC CTGGG GC GAAGTACGTT Ni3290 AAACAGTTCT TGCCCACAAG GCCTGGAGAA CACGATC CTAGG GC GAAGTACGTT NC4832 AGACAGTTCT TGCGCACAAG GCCTGGAGAA CACGATC CTAGG GC GAAGTACGTT NC1130 AGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTGGG GC GAAGTACGCT Ntl270 AGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTGGG GC GAAGTACGCT NC4711 AGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTGGG GC GAAGTACGCT Nd3228 AGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTGGG GC GAAGTACGCT Nt6583 GGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTAGG GC GAAGTACGCT NS5941 GGACAGTTCT CGCGCCCAAA GCCTGGAGAA AGACACGATT CTAGG GC GAAGTACGTT NC4486 AGACAGTTCA GGCGCGTAAG GCCTGGAGAA GGATACGATT CTGGG GC GAAGTACGCT Nd5923 GGAATGTTCG TACCGGCACC ATATGAGGAA AGAGAT TACGTT NC1455 GGA -AAGTTGGTT Nc9S7 GGA -AAGTTGGTT NC1945 GGA -AAGTTGGTT Ndil692 GGA -AAGTATGTT Nd6788 GGA -AAGTTGGTT Nc4499 GGA -AAGTTGGTT 552740 TGGCCCCTAG TCACGGGCAC TTTTGGAGGA Sbl903 TGGCCTTTAG TCACCGGTAC GTTTGGAGGA Sb7140 TGGCCTTTGG TCACGGGTAC TTTTGGAGGA 552741 TGGCCTTTGG TCACGGGTAC TTTTGGAGGG NC4481 TGGCCTCTAG TCACCGGTAC ATTTGGAGGA NC1824 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG Ni3721 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG Gsp8239 TGGCCTTTAG TAACGGGCAC ATTTGGAGGA Gsp8241 TGGCCTTTAG TAACGGGAAC GTTTGGAGGA NS963 TGGCCTTTAG TCACCGGTAC TTTTGGAGGG Sh2739 TGGCCTTTAG TCACCGGTAC ATTTGGAGGG Nd3319 TGGCCTCTAG TTACCGGCAC TTTTGGAGGT Nd6793 TGGCCTTTAG TCACCGGTAC GTTTGGAGGA Gsp8242 TGGCCTCTAG TCACCGGTAC TTTTGGAGGA NC24 89 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG NC4 709 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG NC84 7 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG NC1693 TGGCCTCTAG TCACCGGTAC ATTTGGAGGG Nil940 TGGCCTTTAG TCACCGGTAC TTTTGGAGGG Nil799 TGGCCTTTAG TCACCGGTAC TTTTGGAGGG NS5940 TGGCCTTTAG TCACCGGTAC TTTTGGAGGG Np7221 TGGCCCCTAG TCACGGGCAC TTTTGGAGGA Gsp8243 TGGCCCCTAG TCACGGGCAC TTTTGGAGGA Ni2316 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA Ni3290 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NC4832 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NC1130 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA Ntl2 70 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NC4711 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA Nd3228 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA Nt6583 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NS5941 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NC4486 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA Nd5923 TGGCCCCTGG TCACGGGCAC TTTTGGAGGA NC1455 TGGCCCTTGG TCACGGGTAC TTTTGGAGGG NC967 TGGCCCTTGG TCACGGGTAC TTTTGGAGGG NC1945 TGGCCCTTGG TCACGGGTAC TTTTGGAGGG Ndil692 TGGCCCCTAG TCACGGGCAC TTTTGGAGGA Nd6788 TGGCCTTTGG TCACCGGTAC TTTTGGAGGG NC4499 TGGCCCTTGG TCACGGGTAC TTTTGGAGGG 7.2 DNA sequences of artificial mutant het-c alleles pdl, pd2, pd3, pol and del3 >pdl 3 ' A A T C G A T C T C C A A A A T G C C G T G A C C A G G G C C A A G C G T A C T T T C C T T C T C C A G G C C T T A C G C G C C T G A A C T G T C T A T T C T C G A G T T T G A G T T T A G T C G C A G T G C C A A C G T G G G G A A A G A C T G G A C T A T G C C C G C C C C G C C T T T C T T C C A T A T C G A T T A G G A C C A A T T C G C A G T A G T T A C T G T G G G C A G N A A A G T C C T C C A G G G T A T G A A G G G C C T G T C C C A A G C A C C T C A A G G C C T C G C A C A A G T C A G A C T C C T T G C C T C T G C C A T G A G A G C C A C T G G T A T A C A G A C G A C C G T A A T G G A T G C A A C G C T G C A A G C T G A A A C G G A T A T A G C C G G C A C T T G T G G T G T T C C C A G C C T T G A T G A T G G G C C A A T T C T T C C G T T G G C G A A T A T A A T T C T T C C A T A C C G G T A T T T G G A T T C G A T C T C N A T T T C T G G C G G G G T G T 5 ' >pd2 3 ' A A T C G A T C C T C C A A A A T G C C G T G A C C A G G G C C A A G C G T A C T T A G G C C T T A C G C G C C T G A A C T G T C T A T T C T C G A G T T T G A G T T T A G T C G C A G T G C C A A C G T G G G G A A A G A C T G G A C T A T G C C C G C C C C G C C T T T C T T C C A T A T C G A T T A G G A C C A A T T C G C A G T A G T T A C T G T G G G C A G G A A A G T C C T C C A G G G T A T G A A G G G C C T G T C C C A A G C A C C T C A A G G C C T C G C A C A A G T C A G A C T C C T T G C C T C T G C C A T G A G A G C C A C T G G T A T A C A G A C G A C C G T A A T G G A T G C A A C G C T G C A A G C T G A A A C G G A T A T A G C C G G C A C T T G T G G T G T T C C A G C C T T G A T G A T G G G C C A G T T C T T C C G T T G G C G A T A T A A T T C T T C C A T A C C G G T A T T T G G A T C G A T C T C G A N T T C C T G C G G G G T T G T T A C T G G T C C T C C G T A G T C C T T G G G T 5 ' >pd3 3 ' A A T C G A T C T C C A A A A T G C C G T G A C C A G G G C C A A G C G T A C T T T A C G C G C C T G A A C T G T C T A T T C T C G A G T T T G A G T T T A G T C G C A G T G C C A A C G T G G G G A A A G A C T G G A C T A T G C C C G C C C C G C C T T T C T T C C A T A T C G A T T A G G A C C A A T T C G C A G T A G T T A C T G T G G G C A G G A A A G T C C T C C A G G G T T A T G A A G G G C C T G T C C C A A G C A C C T C A A G G C C T C G C A C A A G T C A G A C T C C T T G C C T C T G C C A T G A G A G C C A C T G G T A T A C A G A C G A C C G T A A T G G A T G C A A C G C T G C A A G C T G A A A C G G A T A T A G C C G G C A C T T G T G G T G T T C C A G C C T T G A T G A T G G G C C A G T T C T T C C G T T G G C G A T A T A A T T C T T C C A T A C C G G T A T T T G G A T C G A T C T C G A G T T C T G C G G G G T T G T T A C T G G T C C T C C G T A A T C C T T G G G T T C C A T A C T C C G C C G G G C A T C C C T T T C C C G T C C A G C C G T T A C C C A A A A G G T T T G T T C G A A T A T G T T C C C T C C G G T T C T T G T T A 5 ' >del3 3 ' A A T C G A C C C T C C A A A A T A C C G T G A C C A A G G G C C A A T C C A T T G A G T G T G A T T C G A G T G G C T G T G C C A A C G T G G G G G A A G A T T C G A C T C T C A G A G C G G G T T T C C T T T T C A T G A A T G T C G A T C A G G G C A A G T T C G C A G T A G T T G C T G T G G G C A G G A A A G T C C T C C A A A G T A T G A A G G G C C T G T C C C A A G C A C C T C A A G G C C T C G C A C A A G T C A G A C T C C T T G C C T C T G C C A T G T G A G C C A C T G G T A T A C A A A C G A C C G T A A T G G A T G C A G C G C T G C A A G C T G A A A C G A A T A T A G C C G G C A C T G G T G G T A T C C C A G C G T T C T C G A T G G G C C A G T T C T T C G T T G G C G A T A T A A T T C T T C A T A C C G G T A T T T G G A T C G A T C T C G A N T T C T G C G G G G T G A A C T G G T C C T C G T A A T C T T G G G T C A T A C T C C G C G G G C A T C C T T T C C A T C C A G C G T A G C C A A N A A G G T T G T C G A A T A T G T T C C T C G G G T C T G T A A A C G C C C A N G C G C T C C T C G G T A A C C T C N A A C T C T T C N G T A G C G T T T C C A T G A A C C A G G A A A G A A A G A A C C C AGAACTANTTAACAAGAATTAGTGGATGGGATGGTACNAAGGCCCGCGAAAAAGGGANGAAAGACTTGCCATGATGCCGGATNGTNACCGCAT T A A C T C C C T T C C T A N C T G C C G A A 5 ' >pol 3 ' A A T C G A T C T C C A A A A T G C C G T G A C C A G G G C C A A G C G T A C T T C G C C C C A G A A T C G T A T C C T T C T C C A G G C C T T A C G C G C C T G A A C T G T C C A T T G T T A C G T G T G T C A T T C T T G A G T G T G A T T C G A G T A T C T G T G C C A A C G T G G G G A A A G A C T G G A C T A T G C C C G C C C C G C C T T T C T T C C A T A T C G A TTAGGACCAACTCGCAGTAGTTACTGTGGGCAGGAAAGACCTCCAGGGTATGAAGGGCCTGTCCCAAGCACCTCAAGGCCTCGCACAGGTCGG A C T C C T T G C C T C T G C C A C G A G A G C C A C T G G T A T A C A G A C G A C C G A A A T G G A T G C A A C G C T C C A A G C T G A A A C G A A T A T A G C C G G C A C T T G T G G T G T T C C A G C C T T G T T G A T A G G C C A A T C T T T C G T T G G C G A T A T A G T T T T T C A T T C C G G A A T A T A A A T C G A T C T C G A G T T C T C C G G G A T G T A C C G G G C C C C G T A G T C T T G G G T C A A A C T C G C G G G C A T C C T T T C C G T C A G C A T A G C C A A G A G G G T T G T C G A T A T G T T C C T C G G G T C T G T A A A C G C C C A G G C G C T C C T C G G T A A C C T C G A A C T C T T C G G T A G C G T A T C C A T G A G C C A G G A A A G A A A G A A C C C A T A C C T A G T G A C A A G A G T T A G T G G A T G G A A T G A A A C G A G G G C A C G G C G A A G A T G G G A C G A A A G A C T T G C A A T G A T G C G A A T G G T A G C C G C A T T A A C T C C C T T C A A G C T G C C G A C A T C G 5 ' 7.3 Sequences of het-c :GFP fusion genes >ORSmGFP 5' C C C A C G C C A T G C C T C T C C G C C A A C A G A T C G C T A C T A C G G C A G C C G C C C C T C T T C C T C T G G G T A T G G A T C G A T C C A A G G A G A T A T A A C A A T G A G T A A A G G A G A A G A A C T T T T C A C T G G A G T T G T C C C A A T T C T T G T T G A A T T A G A T G G T G A T G T T A A T G G G C A C A A A T T T T C T G T C A G T G G A G A G G G T G A A G G T G A T G C A A C A T A C G G A A A A C T T A C C C T T A A A T T T A T T T G C A C T A C T G G A A A A C T A C C T G T T C C A T G G C C A A C A C T T G T C A C T A C T T T C T C T T A T G G T G T T C A A T G C T T T T C A A G A T A C C C A G A T C A T A T G A A G C G G C A C G A C T T C T T C A A GAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACCATCTCTTTCAAGGACGACGGGAACTACAAGACACGTTGCTGAAGTCAAG T T T G A G G G A G A C A C C C T C C G T C A A C A G G A T C G A A C T T T A A G G G A A A T C C A T T T C C A A G G A G G A C G G A A A C A T C C T C C G G C C A C A A T T T G G A A T A C A A C T A C A C T C C C C A C A A C G T T A T A C A T C C A C G G C A G A A C A A A C A A A A G A A T G G A A T C C N A A G C T A A C T T C C A A A A T T T A G A C C C A C A T T G A A N A N T G G A A N C G T T C C A C T A N C A A A C C A T T A T C N A C N A A T A C T C C C A T T N G G G A A G G C C C T G T C C T T T T A C C A A A N A C C A T T T T 3 ' 225 

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