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Gene interactions during Neurospora development Gavric, Olivera 2002

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GENE INTERACTIONS DURING NEUROSPORA DEVELOPMENT by OLIVERA GAVRIC B.Sc , University of Sarajevo, 1987 ATHESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A July 2002 © Olivera Gavric, 2002 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 ^hdjsil/l The University of British Columbia Vancouver, Canada Date | ^ / c | 10J Ifynt DE-6 (2/88) A B S T R A C T There are more than 100 loci that encode products that can affect tip growth and branching in Neurospora crassa. In order to dissect the genetics of branching the functional relationship between 36 of those loci have been examined. By using epistasis analysis some of these genes were grouped into common developmental pathways. Double mutants were obtained through ascus analysis. In most cases double mutants show a phenotype more severe than either single mutant alone, indicating that each mutant probably affects separate pathways involved in hyphal tip growth and branching. Several double mutants were found to closely resemble one of the parental strains in hyphal morphology. This suggests both genes are part of a single developmental pathway, with the epistatic gene acting before the hypostatic one. However, about half of the double mutants obtained in this study did not fit the traditional view of epistasis. The presented analysis gave a provisional characterization of four pathways involved in tip growth and branching. In addition, all these morphogenes were phenotypicaly characterized and screened for their response to exogenous C a 2 + and M n 2 + . Mutant phenotypes of snowflake, smco-1 and smco-6 were corrected to wild type phenotype either by addition of exogenous C a 2 + or M n 2 + . Some mutants were partially corrected by only C a 2 + or M n 2 + . Also, growth of several mutants was inhibited either by C a 2 + or M n 2 + o r both. i i TABLE OF CONTENTS Abstract i i List of Tables vi List of Figures • vii Acknowledgements xvii CHAPTERI 1 1. G E N E R A L INTRODUCTION 1 1.1. Cell Components Involving Hyphal Tip Growth and Branching 2 1.2. Models of Hyphal Tip Growth and Branching 9 1.3. Signaling Pathways Involved in Tip Growth 11 1.3. l.Ca2 +Signaling 12 1.3.2. cAMP Signaling 13 1.4. Morphogenes 14 1.5. Epistasis Analysis 15 1.6. Objectives 17 CHAPTER II 18 2. Phenotypic Characterization of Selected Morphological Mutants 18 2.1. Introduction 18 2.2. Materials and Methods 20 2.2.1. Fungal Strains 20 2.2.2. Growth Analysis 21 2.3. Results 22 2.3.1. Phenotypic Description of Morphological Mutants 22 2.4. Discussion 36 2.4.1. Vegetative Hyphae 36 2.4.2. Aerial Hyphae and Conidiogenesis 37 2.4.3. Sexual Structure 38 2.4.4. Mutants That Did Not Fit Assigned Categories 38 ii i CHAPTER III 60 3. Epistatic Interactions among Selected Morphological Mutants of 60 Neurospora crassa 3.1. Introduction 60 3.2. Material and Methods 61 3.2.1. Octads Analysis 61 3.2.2. Measurement of Growth 62 3.2.3. Random Ascospore Analysis 63 3.2.4. Heterokaryon 63 3.3. Results 63 3.3.1. Crosses 63 3.3.2. Phenotypic Characterization of Double Mutants 65 3.3.2.1. Group of Double Mutants Showing Full Epistasis 65 3.3.2.2. Group of Double Mutants Showing Partial Epistasis 68 3.3.2.3. Group of Double Mutants Showing Coepistasis 72 3.3.2.4. Group of Double Mutants Called Novel 75 3.3.2.5. Group of Double Mutants Showing Cumulative Effect 78 of Both Mutations 3.3.3. Grouping of Morphogenes into Common Developmental 81 Pathways 3.4. Discussion 82 3.4.1. Crosses 82 3.4.2. Possible Reasons for the Complexity of Interactions 84 3.4.2.1. Residual Expression of Hypostatic Gene 84 3.4.2.2. Pleiotropy 85 3.4.3. Double Mutants Resembling Non-parental Morphological 86 Mutants 3.4.4. Provisional Characterization of Three Pathways Involved 87 in HyphalTip Growth and Branching iv 3.4.5. Interesting Interactions of cot-1 Mutant 89 CHAPTER IV 128 4. Response of Morphological Mutants to Exogenously 128 Added C a 2 + and M n 2 + 4.1. Introduction 128 4.2. Material and Methods 128 4.3. Results 129 4.4. Discussion 131 4.4.1. Response of Single Mutants to Exogenous C a 2 + and M n 2 + 131 4.4.2. Response of Double Mutants to Exogenous C a 2 + and M n 2 + 132 CHAPTER V 147 5. Integrated Discussion 147 5.1. Future Studies 148 Bibliography 150 Appendices 158 Table 13 Attempted crosses 159 Morphology of colony and hyphae Figures 83-159 164 v LIST OF TABLES Table 1 Morphological mutant used in this study Table 2 Summary of quantitative and qualitative characterization of morphological mutant phenotypes on Vogel's medium at 25 °C. Table 3 Summary of qualitative and quantitative characterization of double mutants showing epistasis. Table 4 Summary of qualitative and quantitative characterization of double mutants showing partial epistasis. Table 5 Summary of quantitative and qualitative characterization of double mutants showing coepistasis Table 6 Summary of quantitative and qualitative characterization of double mutant showing a novel phenotype Table 7 Summary of quantitative and qualitative characterization of double mutants showing cumulative effect of both mutations Table 8 Double mutants showing full and partial epistasis Table 9 Conidiation of double mutants and their single mutant parents Table 10 Growth rates of double mutants (dm) and their single mutants parents on Vogel's minimal medium Table 11 Response of morphological mutants to 100 m M Ca and 5 m M Mn' 2+ Table 12 Morphological mutants that did not change morphology either on 100 m M C a 2 + or 5 m M M n 2 + or both. Table 13 Attempted crosses between morphological mutants 20 35 66 69 72 76 79 82 91 94 130 130 159 vi L I S T O F F I G U R E S Figure 1 Morphology of colony and hyphel of wild type 41 Figure 2 Morphology of colony and hyphae of col-1 42 Figure 3 Morphology of colony and hyphae of col-4 42 Figure 4 Morphology of colony and hyphae of col-8 43 Figure 5 Morphology of colony and hyphae of col-12 43 Figure 6 Morphology of colony and hyphae of col-15 44 Figure 7 Morphology of colony and hyphae of col-16 44 Figure 8 Morphology of colony and hyphae of col-17 45 Figure 9 Morphology of colony and hyphae of smco-1 45 Figure 10 Morphology of colony and hyphae of smco-4 46 Figure 11 Morphology of colony and hyphae of smco-5 46 Figure 12 Morphology of colony and hyphae of smco-6 47 Figure 13 Morphology of colony and hyphae of smco-7 , 47 Figure 14 Morphology of colony and hyphae of smco-8 48 Figure 15 Morphology of colony and hyphae of smco-9 48 Figure 16 Morphology of colony and hyphae of spco-4 49 Figure 17 Morphology of colony and hyphae of spco-5 49 Figure 18 Morphology of colony and hyphae of spco-6 50 Figure 19 Morphology of colony and hyphae of spco-7 50 Figure 20 Morphology of colony and hyphae of spco-9 51 Figure 21 Morphology of colony and hyphae of spco-10 51 Figure 22 Morphology of colony and hyphae of spco-11 52 Figure 23 Morphology of colony and hyphae of spco-12 52 vii Figure 24 Morphology of colony and hyphae of frost 53 Figure 25 Morphology of colony and hyphae of gran 53 Figure 26 Morphology of colony and hyphae of spray 54 Figure 27 Morphology of colony and hyphae of peak 54 Figure 28 Morphology of colony and hyphae of snow flake (sn) 55 Figure 29 Morphology of colony and hyphae of cot-1 55 Figure 30 Morphology of colony and hyphae of ipa 56 Figure 31 Morphology of colony and hyphae of gna-1 56 Figure 32 Morphology of colony and hyphae of curly (cy) 57 Figure 33 Morphology of colony and hyphae of scr 57 Figure 34 Morphology of colony and hyphae of crisp (cr-1) 58 Figure 35 Morphology of colony and hyphae of mcb 58 Figure 36 Morphology of colony and hyphae of delicate (del) .. 59 Figure 37 Morphology of colony and hyphae of coil 59 Figure 38 Provisional pathways involved in vegetative hyphae growth 82 Figure 39 Morphology of colony and hyphae A: col-8, 97 B: spco-11;col-8, C: spco-11 Figure 40 Morphology of colony and hyphae A : pk, 98 B: smco-8;pk, C: smco-8 Figure 41 Morphology of colony and hyphae A: col-5, 99 B: spco-1 l;col-15, C: spco-11 Figure 42 Morphology of colony and hyphae A: col-16, 100 B: col-16;fr, C:fr Figure 43 Morphology of colony and hyphae A:fr, 101 B: smco-5 fr, C: smco-5 Figure 44 Morphology of colony and hyphae A: smco-7, 102 B: smco-7;gna-1, C: gna-1 viii Figure 45 Morphology of colony and hyphae A: spco-10, B: spco-1 l;spco-10, C: spco-11 Figure 46 Morphology of colony and hyphae A: fr, B: smco-8 fr, C: smco-8 Figure 47 Morphology of colony and hyphae A: fr, B: scr;fr, C: scr Figure 48 Morphology of colony and hyphae of A: col-4, B: col-4;col-17, C: col-17 Figure 49 Morphology of colony and hyphae A: smco-7, B: smco-7;gran, C: gran Figure 50 Morphology of colony and hyphae A: col-16, B: col-16;mcb, C: mcb Figure 51 Morphology of colony and hyphae A: col-16, B: col-16;col-4, C: col-4 Figure 52 Morphology of colony and hyphae A: spco-6, B: spco-6;gran, C: gran Figure 53 Morphology of colony and hyphae A: smco-6, B: smco-6;fr,C: fr Figure 54 Morphology of colony and hyphae A: smco-8, B: smco-8';col-15,C: col-15 Figure 55 Morphology of colony and hyphae A: spco-11, B: spco-1 l;gna-l, C: gna-1 Figure 56 Morphology of colony and hyphae A: gran, B: gran;spco-12, C: spco-12 Figure 57 Morphology of colony and hyphae A: col-16, B: col-16;gran, C: gran Figure 58 Morphology of colony and hyphae A: spco-4, B: spco-4;col-l, C: col-1 Figure 59 Morphology of colony and hyphae of A: smco-7, B: smco-7;fr, C: fr Figure 60 Morphology of colony and hyphae A: cot-1, B: cot-l;fr, C: fr ix Figure 61 Morphology of colony and hyphae A: smco-8, 119 B : smco-8;col-4, C: col-4 Figure 62 Morphology of colony and hyphae A: smco-8, 120 B: smco-8;smco-7, C: smco-7 Figure 63 Morphology of colony and hyphae A: smco-8, 121 B: smco-8;cot-l, C: cot-1 Figure 64 Hyphal morphology of wild type A: vegetative hyphae, 122 B: aerial hyphae Figure 65 Hyphal morphology offrost A : vegetative hyphae, 123 B: aerial hyphae Figure 66 Hyphal morphology of spco-4 Arvegetative hyphae, 124 B: aerial hyphae Figure 67 Hyphal morphology of col-17 A : vegetative hyphae 125 B: aerial hyphae Figure 68 Hyphal morphology of the double mutant cot-1, fr 126 A : vegetative hyphae, B: aerial hyphae Figure 69 Hyphal morphology of smco-7 A: vegetative, D: aerial; 127 smco-7;gna-l B: vegetative, E: aerial; gna-1 C: vegetative, F: aerial Figure 70 Response of sn to exogenous C a 2 + and M n 2 + 134 Figure 71 Response of smco-1 to exogenous C a 2 + a n d M n 2 + 135 Figure 72 Response of smco-6 to exogenous C a 2 + and M n 2 + 136 Figure 73 Response of col-15 to exogenous C a 2 + and M n 2 + 137 Figure74 Response of col-12 to exogenous C a 2 + and M n 2 + 138 Figure 75 Response of smco-8 to exogenous C a 2 + a n d M n 2 + 139 Figure 76 Response of gran to exogenous C a 2 + and M n 2 + 140 Figure 77 Response of spco-11 to exogenous C a 2 + and M n 2 + 141 Figure 78 Response of col-17 to exogenous C a 2 + and M n 2 + 142 Figure 79 Response of wild type to exogenous Ca 2 + and M n 2 + 143 x Figure 80 Response of the double mutant gran;fr to exogenous 144 C a 2 + and M n 2 + Figure 81 Response of the double mutant smco-8;fr to exogenous 145 C a 2 + and M n 2 + Figure 82 Response of the double mutant cot-l;fr to exogenous 146 C a 2 + and M n 2 + Figure 83 Morphology of colony and hyphae A: col-15, 164 B: spco-4;col-15, C: spco-4 Figure84 Morphology of colony and hyphae A: col-15, 165 B: col-15;smco-7, C: smco-7 Figure 85 Morphology of colony and hyphae A: fr, 166 B: col-4;fr, C: col-4 Figure 86 Morphology of colony and hyphae A: spco-12, 167 B: col-4;spco-12, C: col-4 Figure 87 Morphology of colony and hyphae A: col-8, 168 B : smco-6;col-8, C: smco-6 Figure 88 Morphology of colony and hyphae A: cr-1, 169 B: smco-7;cr-l, C: smco-7 Figure 89 Morphology of colony and hyphae A : col-15, 170 B: col-15;gna-l, C: gna-1 Figure 90 Morphology of colony and hyphae A: cr-1, 171 B: mcb;cr-l, C: mcb Figure 91 Morphology of colony and hyphae A: col-16, 172 B: col-16 gna-1, C: gna-1 Figure 92 Morphology of colony and hyphae A:fr, 173 B: ipafr, C: ipa Figure 93 Morphology of colony and hyphae A: smco-7, 174 B: smco-7;mcb, C: mcb Figure 94 Morphology of colony and hyphae A: fr, 175 B: coil;fr, C: coil Figure 95 Morphology of colony and hyphae A: spco-6, 176 B: col-16;spco-6, C: col-16 xi Figure 96 Figure 97 Figure 98 Figure 99 Figure 100 Figure 101 Figure 102 Figure 103 Figure 104 Figure 105 Figure 106 Figure 107 Figure 108 Figure 109 Figure 110 Morphology of colony and hyphae A: spco-4, B: spco-4;col-17, C: col-17 Morphology of colony and hyphae A: col-4, B: col-4;gran, C: gran Morphology of colony and hyphae A: fr, B: spco-4;fr, C: spco-4 Morphology of colony and hyphae A: col-4, B: col-4;spco-9, C: spco-9 Morphology of colony and hyphae A: fr, B: gran;fr, C: gran Morphology of colony and hyphae A: fr, B: mcb;fr, C: mcb Morphology of colony and hyphae A: fr, B: spco-6;fr, C: spco-6 Morphology of colony and hyphae A: smco-7, B: spco-4;smco-7, C: spco-4 Morphology of colony and hyphae A: spco-4, B: spco-4;gna-1, C: gna-1 Morphology of colony and hyphae A: col-4, B: col-4; spco-10, C: spco-10 Morphology of colony and hyphae A: smco-7, B: smco-7;col-17, C: col-17 Morphology of colony and hyphae A: col-15, B: col-15;spco-9, C: spco-9 Morphology of colony and hyphae A: col-16, B: col-16;spco-10, C: spco-10 Morphology of colony and hyphae A: col-4, B: col-4;gna-l, C: gna-1 Morphology of colony and hyphae A: col-16, B: col-16;smco-l, C: smco-1 xii Figure 111 Morphology of colony and hyphae A: col-16, B: col-16; smco-5, C: smco-5 Figure 112 Morphology of colony and hyphae A: spco-11, B: spco-11;gran, C: gran Figure 113 Morphology of colony and hyphae A: cot-1, B: cot-1;gran, C: gran Figure 114 Morphology of colony and hyphae A: smco-7, B: smco-7;col-4, C: col-4 Figure 115 Morphology of colony and hyphae A: gran, B: spco-4;gran, C: spco-4 Figure 116 Morphology of colony and hyphae A: cot-1, B: smco-7;cot-1, C: smco-7 Figure 117 Morphology of colony and hyphae A : col-4, B: col-4 cot-1, C: cot-1 Figure 118 Morphology of colony and hyphae A: col-15, B: col-15;spco-7, C: spco-7 Figure 119 Morphology of colony and hyphae A: cot-1, B: spco-4;cpt-l, C: spco-4 Figure 120 Morphology of colony and hyphae A: col-17, B : smco-5;col-l 7, C: smco-5 Figure 121 Morphology of colony and hyphae A : cot-1, B: cot-1 ;col-16, C: col-16 Figure 122 Morphology of colony and hyphae A: gran, B: gran;col-1, C: col-1 Figure 123 Morphology of colony and hyphae A: spco-4, B: spco-4;pk, C: pk Figure 124 Morphology of colony and hyphae A: col-16, B: col-16;spco-5, C: spco-5 Figure 125 Morphology of colony and hyphae A: smco-9, B: spco-6; smco-9, C: spco-6 Figure 126 Morphology of colony and hyphae A: smco-9, B: smco-9;col-l6, C: col-16 xiii Figure 127 Morphology of colony and hyphae A: cr-1, B: cr-1;cot-1, C: cot-1 Figure 128 Morphology of colony and hyphae A: col-15, B: col-15 col-16, C: col-16 Figure 129 Morphology of colony and hyphae A: smco-6, B: smco-6;col-l6, C: col-16 Figure 130 Morphology of colony and hyphae A smco-7, B: smco-7;col-l6, C: col-16 Figure 131 Morphology of colony and hyphae A:smco-8, B: smco-8 spco-4, C: spco-4 Figure 132 Morphology of colony and hyphae A: gran, B: gran;cy. C: cy Figure 133 Morphology of colony and hyphae A: spco-11, B: spco-11; col-1, C: col-1 Figure 134 Morphology of colony and hyphae A: spco-4, B : spco-4;mcb, C: mcb Figure 135 Morphology of colony and hyphae A: col-15, B: col-15;spco-9, C; spco-9 Figure 136 Morphology of colony and hyphae A: smco-6, B: smco-6;col-l7, C: col-17 Figure 137 Morphology of colony and hyphae A: smco-6, B: smco-6;col-l5, C: col-15 Figure 138 Morphology of colony and hyphae A: cot-1, B: cot-1;gna-1, C; gna-1 Figure 139 Morphology of colony and hyphae A : mc,b B: mcb;cot-l, C: cot-1 Figure 140 Morphology of colony and hyphae A: spco-4, B: spco-4;col-4, C: col-4 Figure 141 Morphology of colony and hyphae A: spco-11, B: col-4;spco-11, C: col-4 Figure 142 Morphology of colony and hyphae A: coU8, B: col-8;col-17,C: col-17 Figure 143 Morphology of colony and hyphae A: smco-6, B: smco-8;smco-6, C: smco-8 Figure 144 Morphology of colony and hyphae A: spco-5, B: smco-8;spco-5, C: smco-8 Figure 145 Morphology of colony and hyphae A: pk, B: col-16;pk, C: col-16 Figure 146 Morphology of colony and hyphae A: sn, B:sn;fr,C:fr Figure 147 Morphology of colony and hyphae A: cot-1, B: cot-1;col-17, C: col-17 Figure 148 Morphology of colony and hyphae A: spco-12, B: spco-12;fr, C: fr Figure 149 Morphology of colony and hyphae A: col-4, B: col-4;spco-6, C: spco-6 Figure 150 Morphology of colony and hyphae A: smco-6, B: smco-6;col-4, C: col-4 Figure 151 Morphology of colony and hyphae A: col-4, B: col-4;spco-5, C: spco-5 Figure 152 Morphology of colony and hyphae A: col-15, B: col-15;fr, C: fr Figure 153 Morphology of colony and hyphae A: sp, B:sp;fr,C:fr Figure 154 Morphology of colony and hyphae A : spco-11, B: spco-11;col-17, C: col-17 Figure 155 Morphology of colony and hyphae A : smco-4, B: smco-4;col-17, C: col-17 Figure 156 Morphology of colony and hyphae A: col-4, B: col-4;smco-l, C: smco-1 Figure 157 Morphology of colony and hyphae A: gran, B: gran;col-17, C: col-17 Figure 158 Morphology of colony and hyphae A: smco-6, B: smco-6;col-l, C: col-1 xv Figure 159 Morphology of colony and hyphae A: col-16, 240 B: smco-4;col-16, C: smco-4 ACKNOWLEDGMENTS I would like to thank my supervisor Dr. Griffiths for his support and my family for their patience. M y thanks to Aleksandra Virag and Eduardo Jovel for their help and Mladen Markovic for his technical expertise. xvii C H A P T E R I 1. GENERAL INTRODUCTION Neurospora crassa, like other filamentous fungi, grows by tubular filaments, hyphae, which are incompletely compartmentalised by septa. Growth of filamentous fungi is highly localized to the terminal portion of the hypha, the apex. This is an extreme example of polarized growth. The process of tip growth is considered the hallmark of the fungal kingdom. Although tip growth occurs in other systems, it is restricted to a few specialized cell types, such as pollen tubes and root hairs (Heath, 1990). Tip growth and branching provide a mechanism for the colony to grow exponentially. Branching can take place either laterally, by the emergence of a new branch along the lateral hyphal walls, or apically, by splitting of the growing apex into two or more apices. The new branch is actually a new hyphal tip and it utilizes the same extension mechanism as hyphal tip growth. The phenomenon of tip growth was recognized over a hundred years ago by Ward (1888). Since then hyphal tip growth has been an attractive subject for experimental and theoretical studies. Important growth events occur within 5 (im of the tip (Heath, 1990). Although many components involved in tip growth have been identified, hyphal tip growth remains poorly understood at present due to its complexity and the difficulty of studying such a restricted area. Tip growth has been extensively studied; in contrast, branching has received relatively little attention. While data about the components involved in hyphal tip growth accumulate (see below), we still lack knowledge of which component or components initiate cellular signals for a new branch. To gain a better understanding of branching and hyphal tip growth I examined 36 morphological mutants. I tried to group those mutants by phenotypic description (Chapter 1 II), by functional interactions, epistasis (Chapter III) and finally by their response to exogenous C a 2 + and M n 2 + (Chapter IV). 1.1. Cell Components Involving Hyphal Tip Growth and Branching The fungal cell wall appears to play an important role in the determination of hyphal morphology. Wild type mycelia of N. crassa form osmotically sensitive, spherical protoplasts when treated with carbohydrases that hydrolyze much of the cell wall polymers. Treatment with lower concentrations of these enzymes causes wild type to assume a very restricted colonial form of growth (Bachman and Bonner, 1959). The major components found in fungal cell walls are glucan (P—1,3-glucan, (}-l,6-glucan) a—chitin and peptide material (Wrathall and Tatum, 1974). Alterations in the relative proportions of any of the hyphal wall components are associated with abnormal growth patterns in N. crassa. The cell walls from wild type N. crassa grown in the presence of the sugar sorbose have an altered chitin to glucan ratio (de Terra and Tatum, 1961) and reduced amount of p—1,3-glucan (Mahadevan and Tatum, 1965) due to inhibition of the enzyme glucan synthase. The most characteristic shape-determining component of fungal walls is chitin, the a- (1-4) —linked homopolymer of N-acetylglucosamine. In N. crassa two chitin synthase genes have been cloned, chs-1 (Yarden and Yanofsky, 1991) and chs-2 (Din and Yarden, 1994). Also, a gene affecting p-glucan synthase activity, gs-1, has been cloned in N. crassa (Enderlin and Selitrnnikoff, 1994). The mutant gs-1 is defective for cell wall formation and P- 1,3-glucan synthase activity. A key feature of hyphal tip growth is the long-distance transport of vesicles containing cell wall precursors to the apex where wall formation occurs. It has been proposed that motor proteins are involved in these processes (Plamann et al., 1994; Bruno et al., 1996; 2 Seiler et al., 1997). Two classes of microtubule associated motors, kinesin and dynein/dynactin, were identified in fungi. Mutations in genes encoding subunits of these motor proteins affect diverse processes such as nuclear migration in the yeast Saccharomyces cerevisiae (Xiang, 1994), nuclear distribution and hyphal morphogenesis in N. crassa (Plamann et al., 1994) and hyphal extension and pathogenicity in Ustilago maydis (Lehmler et al., 1997). Cytoplasmic dynein is a large, heteromultimeric protein that requires another heteromultimeric protein called dynactin to transport cargo. Mutation in any subunit of the N. crassa dynein/dynactin complex has the same phenotypic effect; eight different loci define a group of phenotypically identical mutants known as ropy (ro). A l l of them are defective in hyphal growth and nuclear distribution, and have a colonial phenotype (Garnjobst and Tatum, 1967; Bruno et al. 1996). In addition to the cytoplasmic dynein/dynactin complex, the kinesin class of microtubule-associated motors has been found in fungi. Kinesin is a plus-end directed motor that is thought to translocate organelles along microtubules in the direction opposite from the dynein-activated movement. Elimination of kinesin in N. crassa, in the mutant nkin, reduces hyphal growth but does not stop it; it also has little or no effect on the movement of organelles (Seiler et al., 1997). The most significant change observed in the mutant concerns the Spitzenkorper. Instead of a single Spitzenkorper, in mutant hyphal tips, a multitude of growth zones exist, leading to profuse branching and bulging of hyphae. This suggests a role of nkin in the transport of small, secretory vesicles. Interestingly, double mutants lacking activity of both dynein/dynactin and kinesin are still viable (Seiler et al., 1997). This probably suggests that other motor proteins are present in N. crassa. 3 The recent study of Lopez-Franco and Bracker (1996) described the Spitzenkorper as a heterogenous, multicomponent, pleomorphic complex without a membrane. It usually consists of an outer vesicle cloud and an inner core; both of which contain small cell components such as microfilaments, microtubules and microvesicles. The same study showed that the organization of Spitzenkorper varies among different fungi. They described eight morphological patterns of Spitzenkorper organization in the higher fungi based on the shape and distribution of their components. It seems that the Spitzenkorper patterns are conserved at the genus level. The Spitzenkorper is a structure ubiquitous in septate fungi undergoing hyphal morphogenesis. Its position is controlled by the cytoskeleton and it is absent in non-growing hyphae, irrespective of how growth is inhibited (Riquelme et al., 1998, 2000). A slight alteration of the position of the Spitzenkorper at the tip during growth is correlated with the local direction of growth (Bartnicki-Garcia et al., 1995). Studies on Aspergillus niger showed that during branch initiation the Spitzenkorper did not divide and instead a new Spitzenkorper appeared by condensation of the vesicle cloud in the vicinity of the branching site (Reynaga-Pena et al., 1997). Although there is substantial amount of evidence linking the Spitzenkorper with the growth of a hyphae of higher fungi, the absence of a Spitzenkorper in the hyphae of lower fungi (Grove and Bracker, 1970) and in other tip growing cells raise the question of whether the Spitzenkorper really plays a crucial role in tip growth. The work of Requelme et al. (1998) implicated the microtubular cytoskeleton in the positioning and movement of the Spitzenkorper in hyphae of N. crassa. Many studies showed that microtubules participate in the transport of secretory vesicles to the hyphal apex (Heath, 1987, 1994). Anti-tubulin drugs applied to plant tip-growing cell types either 4 had no effect on tip growth (Kropf et al., 1998) or caused growth inhibition (Bibikova, 1999). While the role of the microtubule cytoskeleton in tip growth is imperfectly understood, it is clear that actin plays important role in tip growth of fungi and tip growing plant cells. Apically growing plant cells treated with actin disrupting drugs immediately show inhibition of growth (Kropf et al., 1998). Filamentous actin (F-actin) is concentrated in the apices of a variety of tip-growing organisms. It is found in fungi (Heath, 1990), the oomycete Saprolegnia ferax (Jackson and Heath, 1993 b), pollen tubes (Picton and Steer, 1982), algal rhizoids (Kropf et al., 1989), and bryophytes (Doonan et a l , 1988) indicating that it plays a general role in tip extension. Actin is one of the most ubiquitous and widely studied proteins found in living cells. Actin occurs as a globular subunit, G-actin that reversibly polymerizes into a 7-nm thick long filament F-actin. Both F and G actin can bind directly, or indirectly via actin binding proteins to diverse cellular structures (Heath, 1994). In Saccharomyces cerevisiae actin is the major cytoskeletal component that provides the structural basis for growth polarity (Bretscher et al., 1994). In contrast to S. cerevisiae, little is known regarding the molecular mechanisms controlling growth polarity of filamentous fungi. The actin gene has been cloned from N. crassa (Tinsley et al.1998). Picton and Steer (1982) suggested that actin plays a direct role in regulation of the shape of tip-growing cells by controlling the extensibility of the tip. The observed apical actin caps of S. ferax and other oomycetes (Heath, 1987, Heath and Kaminskyj, (1989) supported this hypothesis. Heath and Kaminskyj proposed that the actin cap in S. ferax regulates tip morphogenesis and it is also implicated in the transport of wall vesicles to the apex. The disruption of the F actin cap by U V microirradiation and treatment with cytochalasin E induced rapid changes in the actin cap (Jackson and Heath, 1990). Cap disruption was accompanied by a transient growth rate increase and subsequent rounding and swelling of apices and bursting of hyphal tips. This indicates that F-actin resists turgor pressure in expanding regions of the hyphae where the cell wall is weak. Based on all these observations Jackson and Heath suggested that the actin cap stabilizes the plastic hyphal tip and localizes vesicle exocytosis. The actin cap is formed at the initiation of branching tips and it is disorganized upon cessation of growth (Heath, 1987). Although the actin cap is universal in the oomycetes (Jackson and Heath, 1990) it has not been reported in most fungi and other tip growing cells. However, the F actin at the tips of these other growing cells forms plaques (Heath, 1990). Numerous studies have showed involvment of C a 2 + ions in the growth of many tip-growing organisms. It seems that the tip-high C a 2 + gradient is a general characteristic of polarized cell growth, irrespective of organismal complexity. A tip-high C a 2 + gradient is found in N. crassa (Shimid and Harold, 1988), S. ferax (Jackson and Heath, 1989; Hyde and Heath, 1997), amoebae and slime moulds (Taylor, 1973; Allen, 1978; Ueda, 1978) pollen tubes (Malho et al., 1996), root hairs of higher plants (Bibikova and Gilroy, 1999, 2000), and fucoid algae (Kropf and Quatrano, 1987; Brownlee andWood, 1986). Several studies have reported morphological effects in oomycetes and fungi in response to disturbances of Ca 2 + . When mycelia of S. ferax (Jackson and Heath, 1989) and N. crassa (Shimid and Harold, 1988) were grown in medium containing low concentrations of Ca2 +ions the rate of tip extension was reduced markedly. Wild type hyphae of N. crassa could be induced to a hyperbranching morphology using the calcium-channel blocker, verapamil (Dicker and Turian, 1990). Further evidence for a role for calcium ions in branch formation comes from experiments with ionophores. In N. crassa, 0.1 m M ionophore stimulated branching (Reissig and Kinney, 1983). In addition, the ./V. crassa hyperbranching mutants frost and spray could be corrected to a wild type branching pattern by the addition of high concentrations (50-6 500 mM) of Ca 2 (Dicker and Turian, 1990; Sone and Griffiths, 1999). Although all tip-growing organisms have a tip-high calcium gradient it seems that the gradient is not maintained in the same way. In some tip-growing organisms such as the oomycete S. ferax, a high C a 2 + gradient in the hyphal tips is generated by C a 2 + influx across the plasma membrane via a tip high gradient of stretch activated channels (Lew, 1999; Garril et al., 1992). Application of gadolinium (Gd3 +) inhibited stretch-activated channel activity and stopped tip growth. The calcium gradient returned and tips growth resumed when G d 3 + was washed out. In contrast, in N. crassa influx of C a 2 + via the stretch-activated channels is not essential for tip growth and stretch-activated channels are not clustered at the tips (Lew, 1999; Silverman-Gavrila and Lew, 2000). Blocking these channels with gadolinium did not disrupt the tip-high cytosolic Ca gradient and only transiently inhibited hyphal tip growth (Levina et al., 1995). This indicates that influx of Ca2 +ions from the medium does not play a major role in the generation of the C a 2 + gradient or the regulation of tip growth in N. crassa. Instead, there is evidence that the C a 2 + gradient is generated internally by C a 2 + sequestration into the endoplasmic reticulum behind the tip and C a 2 + is released from tip-localized vesicles via an IP3 receptor activated by tip-localized IP3. The location of IP3 is maintained by the actin cytoskeleton (Harold and Harold, 1986). While data about involvment of Ca ions in hyphal tip growth and branching in filamentous fungi accumulate, relatively little is known about the role of M n 2 + ions. Manganese is one of the essential trace elements. Studies in S. cerevisiae showed that calcineurin is required for the tolerance to high concentrations of manganese and it is the factor that regulates manganese uptake (Farcasanu et al. 1995). Loukin and Kung, (1995) proposed the role of M n 2 + as an intracellular transducer. According to these authors, it was established that M n 2 + behaived similar to C a 2 + in its ability to bind to cell receptors 7 with intermediate affinity. The same authors showed that M n was much more efficient than C a 2 + in rescuing cell cycle arrest of the mutant cdcl. This may suggest the role of M n 2 + ions in cell-cycle progression. 1.2. Models of Hyphal Tip Growth and Branching Old models advocate turgor as a force that exerts pressure on the deformable hyphal tip, which in some way adds wall material as the tip expands. One version of those models was proposed by Bartnicki-Garcia (1973). According to this model, cell wall growth involves a dynamic balance between enzymes lysing walls and those synthesizing them; extensibility of the mature cell wall is regained by supplying adequate softening enzymes to the branch site. In contrast, another model proposed by Wessels (1990) did not involve lytic enzymes. According to this model cell wall extensibility is a product of wall synthesis and the formation of interpolymer cross-linkages that impart tensile strength to the wall. Although Wessels' model is an attractive proposition, wall lytic enzymes such as chitinases and glucanases have been found in actively growing fungal hyphae (Rosenberg, 1979; Gooday and Gow, 1990). Several models proposed that tip growth vesicles are the key elements whose accumulation triggers branching (Trinci, 1974; Katz, et al., 1972; Prosser and Trinci, 1979). The vesicular theory of growth developed by Trinci (1974) suggests that a branch is initiated when the rate of vesicle incorporation and the hyphal growth unit exceed a minimal threshold. The hyphal growth unit is defined as the ratio between the total hyphal length and the total number of hyphal tips. A similar model has been proposed by (Katz et al. 1972) using another ascomycete, A. nidulans. According to this model a new growing point is formed when the capacity of the hyphae to extend exceeds that of the existing growing points. 8 The above models did not account specifically for the shape of the tip but Bartnicki-Garcia dealt with this in detail. In the model proposed by Bartnicki-Garcia (1989), the Spitzenkorper functions as a vesicle supply center (VSC) by regulating the traffic of wall-building vesicles. According to this model the Spitzenkorper is a site for the collection of vesicles from their subapical sources and their subsequent distribution to the wall. Vesicles are released at random in all directions from the V S C and their exocytosis automatically yields the shape of the hyphal tip. According to the same author, a new vesicle supply center is created at each new branching site. The model did not explain how a new Spitzenkorper appears. Although, the role of the Spitzenkorper as a shape-generator is interesting, it does not apply to other tip-growing organisms. Other tip growing cells that do not have Spizenkorpers (pollen tubes and root hairs) also generate a tubular shape. The study of Colling and Trinci, (1974) showed that a large number of vesicles is needed at the apex; over 38, 000 vesicles fuse with the hyphal tip every minute in wild type hyphae of N. crassa. Vesicles carry precursors and enzymes needed for plasma membrane and wall formation to the cell surface. Colling and Trinci (1974) suggested that vesicles are generated throughout the peripheral growth zone of hyphae and they are transported to the apex. Then the imposition of a barrier (e.g. a septum) to acropetal vesicle transport will result in an accumulation of vesicles behind the barrier. Although this model explained the formation of lateral branches it did not address the formation of apical branches. In some fungi, the septum is perforated by a central pore and cell organelles migrate through the pore toward the tip. N. crassa, which has perforated septa, predominantly forms lateral branches. 9 A model proposed by Prosser and Trinci, (1979) gives options for both apical and lateral branching. They suggested that a new branch is initiated either when the flow of vesicles to the extending hyphal tip exceeds the rate at which they can be absorbed, or when a septum reduces or prevents the flow of vesicles to the hyphal tip. Actually, vesicles have been observed behind the plugged septa of N. crassa (Trinci and Collinge, 1974). In addition, the septation mutants of A. nidulans form septa at 25 °C but not at 37 °C (Trinci and Morris, 1979). Growth at 37 °C is characterized by formation of both apical and lateral branches, but at 25 °C only lateral branches are formed. Apical branching at 37 °C could therefore have resulted from transport to the tip of vesicles whose movement would otherwise have been prevented by septa. Although these models that involve septa in branch initiation are attractive, the branching in other fungi is not associated with septation. Rhizopus stolonifer, Astinomucor repens and other members of the Mucorales branch normally, but they are aseptate. In addition, the variety of branching patterns produced by hyphae of N. crassa recovering from an osmotic shock (Trinci and Collinge, 1974) suggests that a branch can potentially be formed from any part of the compartment wall, including the septum. In the model proposed by Walters et al., (1999) branch inititation is partially controlled by determining factors occurring at the previous branching site. This conclusion was based on analysis of branch symmetry. Several models of the regulation of hyphal tip growth have proposed C a 2 + ions as a key component (Picton and Steer, 1982; Jackson and Heath, 1989; Heath, 1995). The evidence in favor of these models is the presence of a tip-high Ca2+gradient in all tip-growing cells. According to the Picton and Steer (1982) model, C a 2 + ions flow into the apex, and a locally elevated level of cytoplasmic C a 2 + stabilizes the fibrillar network composed mainly of microfilaments, which 10 supports the tip region against internal osmotic pressure. Tip extension is promoted by a lowering of the local cytoplasmic C a 2 + ion concentration by sequestering it in the storage vesicles immediately behind the apex, which leads to a weakening of the fibrillar network. Also, this model implicates actin into tip growth. The apical wall of tip growing organisms would be too weak to support the expanding apex and actin may serve this function and thereby control apical extension. Another model proposed by Heath, (1995) also implicates cytoskeleton and calcium in tip extension. According to his model, the cytoskeleton has a prominent role in determining and maintaining the shape of the tip. He proposed that a pheripheral network of F-actin maintains a structured cytoplasm at the tip and maintains connections, via integrin-like molecules embedded in the plasmalemma, with the new wall itself. 1.3. Signaling Pathways Involved in Tip Growth Undoubtedly, all living organisms use signaling mechanisms to regulate transcription and translation on the basis of changes in the extracellular environment. Many studies suggest that the control of hyphal tip growth and branching is inpart achieved through the activation of intracellular communication networks in response to external stimuli. Our knowledge of the components of the cell signalling pathways controlling morphogenesis, to a large extent, comes from studies of pseudohyphal growth of the model organism S. cerevisiae. Pseudohyphal growth of S. cerevisiae requires at least two signaling pathways: the pheromone responsive M A P kinase cascade and the Gpa2-cAMP-PKA signaling pathway. The molecular dissection of morphogenesis in filamentous fungi has been based on the strong evolutionary conservation of fungal signal transduction pathways. 11 1.3.1. Caz+Signaling It is known that numerous cellular processes are under the control of Ca 2 + . Calcium 2+ homeostasis is tightly controlled in all eukaryotic cells since intracellular Ca plays an important role in general signal transduction and in the regulation of many proteins. Many studies in filamentous fungi showed that calcium signaling is involved in hyphal tip growth and branching (Shimid and Harold, 1988; Jackson and Heath, 1989; Dicker and Turian, 1990; Sone and Griffiths, 1999;). Calcium appears to activate calmodulin, a small, highly conserved, Ca2 +-binding protein present in all eukaryotic cells. Calmodulin has been identified and cloned from many organisms including N. crassa (Capelli et al. 1993) . Calmodulin is necessary for normal fungal growth. Disruption of this gene in A. nidulans, (Rassmusen et al., 1994), and S. cerevisiae (Davis et al., 1986) is lethal. Calmodulin is ubiquitous and functions to regulate various membrane proteins and enzymes. One of these enzymes is calcineurin it is a highly conserved calcium-calmodulin regulated serine /threonine phospoprotein phosphatase (Klee and Cohen, 1988). The functional enzyme is a heterotrimer composed of a catalytic subunit (calcineurin A), a regulatory subunit (calcineurin B) and calmodulin (Stemmer and Klee, 9+ 1994) . Calcineurin is the only known protein phosphatase that is dependent upon Ca and calmodulin for the activity. Two calcineurin catalytic subunits cnal and cna2 and regulatory subunit cnbl have been identified and cloned in the yeast S. cerevisiae (Kuno et al., 1991). While the regulatory and catalytic subunits of calcineurin in S. cerevisiae are not essential, disruption of the genes that encode the catalytic subunit in A. nidulans is lethal (Rasmussen et al., 1994). Partial inhibition of calcineurin with antisense R N A to the calcineurin A gene (cna-1), or by administration of cyclosporin A or FK506, caused the disappearance of the tip-high C a 2 + gradient, growth arrest and intense branching in N. 12 crassa (Prokisch, et al., 1997). Also, studies of Kothe and Free, (1998) showed that calcineurin plays a role in signal transduction events that regulate hyphal growth and differentiation in N. crassa. 1.3.2. cAMP Signaling There is much evidence that the cAMP- P K A pathway regulates fungal growth. Several components of this pathway have been identified in filamentous fungi. Heterotrimeric G proteins (afty) are central components of major signaling pathways in eukaryotic organisms (Birnbaumer, 1992). G a subunits have been identified in a number of fungi (i.e. S. cerevisiae, N. crassa, U. maydis). Three G a subunits of the heterotrimeric G protein gna-1, gna-2 and gna-3, which are homologues of G a family, have been cloned in N. crassa (Turner and Borkovich, 1993; Kays, 2000). The gna-1 mutant is defective in apical extension, aerial hyphae development, conidiation and female fertility (Ivey et al. 1996). Similarly, deletion of the gene for the G a subunit gpa-3 in U. maydis (Hartmann et al. 1996) results in altered morphology. Another component from this pathway, adenylyl cyclase, has been identified and cloned in yeast and filamentous fungi. The N. crassa morphological mutant crisp-1 is deficient in adenylyl cyclase, has altered hyphal tip growth and branching and high, constant conidiation. External addition of cAMP corrects the mutant cr-1 phenotype to essentially wild type. This indicates that the cAMP level plays an important role in regulation of the differentiation processes (Terenzi et al., 1974). In addition, Reissing and Kinney (1983) found that the effect of ionophores on branching in N. crassa could be suppressed by exogenous cAMP. Disruption of the adenylate cyclase gene in Ustilago maydis results in constitutive filamentous growth (Gold et al., 1994). 13 There is also evidence that the c A M P - P K A pathway regulates polarized deposition of wall material in hyphae of N. crassa. The mcb gene of N. crassa encodes the regulatory subunit of the cAMP dependent protein kinase A (PKA) (Bruno et al., 1996). Mutation at this locus causes loss of growth polarity when incubated at the restrictive temperature. Protein kinase A catalytic subunits are activated after cAMP induces disssociation of the regulatory subunits from them. The mcb phenotype suggests that cAMP affects growth polarity through protein A activity. Furthermore, mutation of the gene for the S. cerevisiae P K A regulatory subunit (Bsyl) results in an increase in cell size prior to commitment to a new round of cell division, and stimulation of pseudohyphal growth (Gimeno et al., 1992). Other forms of G proteins, small G proteins, Ras2p and Gpa2p have been identified in the yeast, S. cerevisiae (Kubler et al., 1997; Xue et al., 1998) and in N. crassa the ras homologue smco-7 (Kana-uchi, 1977). The ras gene family encodes small G proteins that play an important role in many signal transduction pathways that regulate cell growth and differentiation. Mutation in the N. crassa ras homolog smco-7 causes changes in hyphal growth and reduces the rate of conidial formation. (Kana-uchi, 1997). The S. cerevisiae ras protein Ras2 is involved in morphogenesis; current models suggest that Ras2p can activate both the pseudohyphal M A P K and cAMP pathways. 1.4. Morphogenes Morphogenes supply powerful tools for the dissection of the molecular mechanisms that underly hyphal tip growth and branching. The term morphogene was used for the first time by William Donachie to designate a gene involved in the production of organismal form (referenced by Harold, 1995). Mutation in a morphogene results in an abnormal shape. Numerous morphological mutants have been isolated from many filamentous 14 fungi. Although many mutants with altered growth have been isolated, most of them have not been studied at the biochemical and molecular levels. Our knowledge of genes governing hyphal growth and morphogenesis is still rudimentary. Certainly we can get more insight in the processes involved in hyphal tip growth and branching through molecular characterization of morphological mutants defective in these processes. Hence branching mutants could prove very usuful in elucidating events underlying hyphal tip growth and branching. More than a hundred morphological mutants have been isolated in N. crassa (Garnjobst and Tatum, 1967; Perkins et al.1982). Most of them exhibit increased or altered branching patterns that lead to novel colony morphologies. Certainly there are many more Neurospora morphogenes to be identified. Even then, when all of them have identified we have to map out the pathways in which they work in order to understand the processes that underline morphogenesis. By determining whether or not their individual mutations affect growth and branching processes at common points of action we may gain insight into the regulation of tip growth and branching. Genetic epistasis is one way to assess whether they act in common or different pathways. 1.5. Epistasis Analysis Epistasis is the phenotypic effect of interaction among mutant alleles at multiple loci. Epistasis (Greek for standing over) is gene interaction that results in masking of the mutant phenotype associated with hypostatic gene by a mutation in the epistatic gene. In other words, epistasis refers to functional interaction between nonallelic genes that results in nonadditive gene expression across loci controlling a particular trait (Mather and Jinks, 1971). 15 Epistasis analysis is a powerful genetic tool for assessing whether two genes act in common or parallel pathways. In general, a cross between two distinguishable single mutants affecting the same trait will yield double mutants among other progeny. The phenotype of the double mutant is compared to the parental mutant strains and i f the genes are in different pathways, the double mutant is expected to suffer from cumulative doses of both mutations. If the double mutant has the phenotype of one of the parents, then both genes are expected to be in the same developmental pathway. In addition, epistasis analysis can infer the order of gene action in a pathway by comparison of the phenotype of a double mutant with that of single mutant parents (Avery and Wasserman, 1992). Only the phenotype associated with the epistatic locus will prevail in the double mutant. There are two distinct types of biological pathways, biosynthetic and regulatory, and the rules for interpreting epistasis are reversed depending upon the type of pathway involved. Both types of pathway involve many components, either genes or gene products that act in a definite sequence. Whereas the biosynthetic pathway functions to produce a final product, the regulatory pathway regulates a final gene. Beyond the differences in their overall functions, there are fundamental differences in the functional relationships between the components in each pathway. Each step in a biosynthetic pathway converts one intermediate to another; each step in this pathway has a positive effect on the subsequent step. The intermediate upstream of the first gene accumulates, but the block at this point eliminates everything downstream, including the intermedate that normally accumulates in the downstream mutant. Therefore, in a biosynthetic pathway the upstream gene is epistatic to the downstream gene. In a regulatory pathway each step activates or inactivates a "switch", which is either the gene itself or its product a given step in a regulatory pathway may act either positively or negatively on the subsequent step. In a regulatory pathway a 16 downstream mutation is usually epistatic because any component upstream of it becomes irrelevant. The position of two genes in the same pahway can be determined without detailed knowledge of the nature of the mutations, the pathway, or the molecular mechanism of regulation (Avery and Wasserman, 1992). 1.6. Objectives Since no thorough characterization of the morphological mutants in N. crassa exists in the literature, a ditailed description of the qualitative and quantitative characteristics of their growth was carried out in this study. 1. The primary goal of this study was to identify interacting Neurospora morphogenes and the developmental pathways where they act. 2. The second objective was to identify other genes, in addition to frost and spray involved in C a 2 + and M n 2 + homeostasis. C H A P T E R II 2. Phenotypic Characterization of Selected Morphological Mutants of Neurospora crassa 2.1. Introduction More than a hundred mutants of N. crassa have been identified that display altered hyphal tip growth and branching (Garnjobst and Tatum, 1967; Perkins et al., 1982). The existence of so many mutant loci affecting hyphal tip growth and branching shows that this is classic example of a multigenic complex system. A l l N. crassa morphological mutants were classified by Garnjobst and Tatum, (1967) into six broad categories: true colonial mutants (col), semicolonial mutants (smco), spreading colonial mutants (spco), 17 speading morphological mutants (mo), colonial temperature sensitive mutants (cot) and distinctive morphological mutants. The classification was based on differences in mycelial growth. Phenotypically the most extreme of these mutants are the colonials, which are characterized by a compact colony consisting of highly branched hyphae. Most of the mutants used in this study fall into first three categories, true colonial (col), spreading colonial (spco) and semicolonial categories (smco). According to (Garnjobst and Tatum 1967) true colonial mutants have very dense growth and they stay restricted under, optimal conditions. Spreading colonial mutants begin their growth as colonials but growth does not remain restricted and they slowly spread over the surface, retaining the same hyphal morphology. Semi colonial mutants initially have restricted growth but after a few days flares of faster growing hyphae with more wild type morphology appear, and they spread over the agar surface. Other morphological mutants not called colonial such as frost (fr), spray (sp), snowflake (sn) and gran grow in the spreading colonial manner but they were not grouped with the spreading colonials. Only a few of these mutants have been studied at the biochemical level and their deficiencies documented. The mutation associated with the phenotype frost is known to cause alterations in the enzyme glucose 6-phosphate dehydrogenase (Scott and Tatum, 1970). In addition, the frost mutant is deficient in cAMP but is not correctable by exogenous cAMP (Rosenberg and Pall, 1979). Studies of Dicker and Turien (1990) and Sone and Griffiths (1999) showed that frost could be corrected to wild type by exogenously added Ca 2 + . Another morphological mutant deficient in cAMP is crisp-1 (cr-1). Exogenous cAMP can correct the mutant phenotype of cr-1 to an essentially wild type (Terenzi et al., 1974). Also, Wrathall and Tatum, (1974) showed that all colonial mutants of N. crassa have reduced quantities of cell wall peptides. 18 Several N. crassa genes involved in hyphal tip growth and branching have been cloned and characterized. The gene crisp-1 encodes adenylyl cyclase (Kore-Eda et al., 1991); the gene colonial temperature sensitive cot-1, encodes the member of the protein kinase family (Yarden et al., 1992); the semicolonial, smco-7 gene is a ras homolog (Kana —uchi et al., 1996). The conditional mutant mcb (microcycle blastoconidiation) is defective in growth polarity when grown at restrictive temperature. Cloning and D N A sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP dependent protein kinase P K A , (Bruno et al., 1996). The gene for the a subunit of the heterotrimeric G protein gna-1 has been cloned in N. crassa (Turner and Borkovich, 1993). Mutation at this locus alters hyphal tip growth and branching, conidiogenesis and sexual development. Eight morphological mutants called ropy, belonging to the category of true colonial morphological mutants (Garnjobst and Tatum, 1967), were shown to be defective in specifying subunits of dynein and dynectin molecular motors i.e.genes ro-1, and ro-3 encode the cytoplasmic dynein heavy chain and pi50 g l u e d respectively (Plamann et al., 1994). The Neurospora kinesin gene nkin has been cloned (Seiler et al., 1997). Also, two N. crassa chitin synthase genes have been cloned chs-1 (Yarden and Yanofsky, 1991) and chs-2 (Din and Yarden, 1994). The calcium related frost gene has been cloned and characterized (Sone and Griffiths, 1999); it showed homology to the yeast gene cdcl and is thought to be involved in M n 2 + homeostasis. Cloning and functional analysis of another calcium related gene spray suggested that the product of this gene is located in an organellar membrane, regulating the distribution of C a 2 + via calcineurin (Bok et al., 2001). Molecular characterization of Neurospora morphological mutants shows that morphogenes either encode cellular building blocks, such as chitin, subunits of motor proteins, or genes that encode components of cell regulatory circuitry, such as protein kinases, calcium binding proteins or G proteins. This suggests that 19 regulation of Neurospora development utilizes the same types of genes involved in regulation of development in yeast and in higher organisms. 2.2. Material and Methods 2.2.1. Fungal Strains Neuropora crasa wild type strains 74-OR 8-la (FGSC# 988), 74-OR23-1A (FGSC# 987) and morphological mutant strains listed in Table 1. were obtained from the Fungal Genetic Stock Center (FGSC), Microbiology Department, University of Kansas. The gna-1 mutant was a gift from Dr. C. Borkovich. able 1. Morphological mutants used in this study FGSC name FGSC name FGSC name FGSC name # # # # 7020 col-1; a 3195 spco-7 ;A 3196 spco-7;a 794 gran ;A 1442 col-4; a 4366 spco-9; A 4253 smco-7;A 4066 cot-1 ;a 1177 col-4; A 1384 spco-10;a 1404 smco-8;a 4065 cot-1; A 1401 col-8 ;a 4384 spco-1l;a 8247 smco-8;A 4345 cr-1 a 1376 col-12 A 4383 spco11 A 7365 smco-9;A 826 cr-1 A 3848 col-15 ;a 6947 spco-12 a 3883 ipa A 7453 mcb ;a 3847 col-15 ;A 1363 smco-1 A 3864 scr ;a 7454 mcb ;A 3462 col-16; a 1367 smco-4 ;a 102 fr a 3860 cy a 3461 col-16; A 8245 smco-4;A 103 fr A 3859 cy A 1373 col-17 ;A 1361 smco-5 a 278 pk ;a 3649 coil ;a 1372 spco-4;a 8246 smco-5 A 947 sn a 3648 coil ;A 2233 spco-4 ;A 4531 smco-5; a 507 sn A 4381 del;A 1374 spco-5 ;a 4530 smco-6 ;A 1175 sp ;a gna-l;a 4382 spco-6 ;a 4254 smco-7 ;a 793 gran ;a 20 2.2.2. Growth Analysis Patterns of growth were examined in petri plates of Vogel's minimal medium (Vogel, 1956) at 25 °C. The mutants were observed microscopically using dissecting and compound microscopes for a period of seven days. Growing hyphae were observed with a Zeiss Axioscope microscope. The characteristics like colony density, branching type (lateral or dichotomous), branching interval, angle of branching and presence or absence of conidia was noted. Also macroscopic appearance such as texture, and aerial hyphae of the colonies were noted. A l l the characteristics were described relative to the wild type control. Elongation of individual hyphae is traditionally used as a measurement of the growth rate. This method is generally quite accurate because of hyphal capability to maintain a relatively constant shape during growth. However, for most of morphological mutants with their distorted hyphal morphologies, this method would not be valid. Instead, growth rates of most mutants were obtained by measuring the colony fronts at regular time intervals. Growth rates were measured on petri plate cultures grown on Vogel s minimal medium at 25 °C. Measurements were carried out using eyepiece micrometers. Three colonies of each mutant strain were measured. This was done by measuring 5 diameters of each colony 5 times, with an hour interval between measuring. Hence, to come up with an hour's growth the average of 15 measurements was used. Also for the frost and ipa mutants linear measurements were done on a microscopic scale by observing individual hyphae because of the complicated colony morphologies of these mutants. Spotting cultures were used for observation of aerial hyphae. An aliquot of 1 ml of cool minimal medium was spotted and spread to 2.5 cm on the lid of petri dish. Medium was inoculated and incubated upside-down for 24-48 hours. 21 For photographing hyphal branching patterns, mutant strains were grown between two sheets of cellophane. An autoclaved disk of cellophane was placed on the surface of the agar medium. The inoculum of either conidia or a mycelial block was inoculated on the cellophane. The inocula were covered with another disk of the cellophane and incubated for 24 or 48 hours at 25 °C. The cellophane prevented hyphae from penetrating the medium, making the colony growth more two-dimensional. Hyphae at the colony front were photographed through a Zeiss Axioscope compound microscope on which a Zeiss M35W camera was mounted, using 35 mm T M A X 400 film. 2.3. Results 2.3.1. Phenotypic Descriptions of Morphological Mutants wild type control (Fig. 1). When a spore becomes polarized, a growth tube emerges at one point on the spore surface. This tube extends by tip growth to become a main hypha. At some point primary branches appear. A primary branch also has the potential to grow by tip growth and to give secondary branches, which will in turn give tertiary branches. Neurospora wild type shows clear hyphal size hierarchy. The size of the diameteres of main hypha and its primary secondary and tertiary branches are different. Neurospora crassa utilizes mainly lateral branching, and dichotomous, apical branching occurs rarely. col-1 ( Fig.2). The col-1 mutant formed a very restricted lumpy colony. The colony did not grow more than 1 cm during a 7-day observation. Although the colony was very small, protoperithecia were formed regularly. The mutant had very dense growth invading the medium. The hyphal morphology was delicate; hyphae were very thin with very short distances between the branches. The dichotomous branching was not observed. The col-1 mutant formed a few short aerial hyphae and no conidia. The col-1 mutant has 22 uniform hyphal size and morphology throughout colony. Because of this col-1 is easy to distinguish from other very restricted morphological mutants pk and col-8. col-4 (Fig.3). The col-4 mutant exhibited spreading colonial growth. A difference in size of the main hyphae and their branches was apparent. It formed many aborted branches. The angle of branching was higher than that of the wild type. The dichotomous branching was not more frequent than in the wild type. This mutant formed the same number of protoperithecia as the wild type. The col-4 mutant formed a large number of aerial hyphae. Aerial hyphae were clustered and radially distributed throughout the colony. The col-4 mutant developed small balls of conidia, distributed radially in the direction of growth. The colony showed extensive growth of hyphae beneath the agar surface. A large number of aborted branches, as well as, abundance and position of aerial hyphae. give a unique phenotype to this mutant. col-8 (Fig.4). The col-8 mutant formed a dense, lumpy colony very similar to that of the col-1 mutant. The mutant formed a large number of hyphae beneath the agar surface. The col-8 mutant had fine hyphae with short distances between branches. Flares of thicker hyphae with longer distances between the branches were often observed. Some of those flaring hyphae had a bumpy appearance probably due to aborted branches. The col-8 showed extensive growth of hyphae within the agar. Although the colony was usually less than 1 cm in diameter, protoperithecia were present. Col-12 (Fig.5). Initially the col-12 mutant formed a very restricted colony but, after a few days, flares of faster growing hyphae appeared and the colony escaped from very restricted growth. Sometimes many branches emerged very close to each other at the apex. Hyphae had a bumpy appearance and dichotomous branching was more frequent 23 than in the wild type. The col-12 formed very few short aerial hyphae and conidia formed only in a test tube, and not on a Petri dish. col-15 (Fig.6).The col-15 mutant formed a very dense colony with regional variation of growth pattern. The colony had a three-dimensional appearance with a dark orange center and whitish perimeter. Most of the colony had fine, hyperbranched hyphae; due to very dense growth only hyphal tips were visible. Sometimes leading hyphae appeared with reduced frequency of branching and extended faster but soon they resumed their increased branching. The col-15 mutant formed larger number of dichotomous branches compared to wild type. The col-15 mutant formed a few conidia in a test tube but did not conidiate in a Petri dish. Although the col-15 mutant formed a very restricted colony, usually about 1 centimeter in diameter, protoperithecia were formed regularly in a Petri dish. The col-15 colony can be easily distinguished because of dark orange center, which grows into mound. col-16 (Fig.7.) The col-16 mutant exhibited regional variation with respect to growth pattern. Some parts of the colony had more frequent branching and these parts of the colony appeared denser. The other parts of the colony had a lower frequency of branching and appeared less dense. Initially col-16 formed a restricted colony but growth escaped by flares. Most hyphal tips were blunt and rounded. This may indicate some defect of Spitzenkorper. Very extensive swellings of hyphal tips were present throughout the entire periphery. Hyphal bursts occurred very often at any site along the hyphal length but most of them occurred at the tips. It formed many dichotomous branches, or sometimes multiple branches appeared at the tip. Due to swelling, some banches appeared thicker than their main hyphae. Aerial hyphae were clustered and they formed balls of conidia. These balls of conidia did not disperse easily in water. Main 24 characteristics of col-16 are swollen flaring hyphae, frequent hyphal bursts, dichotomous branching and balls of conidia. col-17 (Fig.8). The hyphal morphology of the col-17 mutant of this mutant was highly irregular with conspicuous swellings. Large swellings were evident at hyphal tips as well as along the entire filaments. Apical and subapical compartments were usually swollen and sometimes three or more apices emerged from the same tip. Many bursts occurred at the hyphal tips; this was more apparent when hyphae were grown between two sheets of cellophane. The angle of branching was larger than the acute angle of the wild type but still not a right angle. Aerial hyphae clustered along the colony perimeter and they were almost absent from the center. Aerial hyphae were longer compared to the wild type but their morphology was totally distorted. Compared to the wild type the col-17 mutant formed fewer amount of conidia. Like col-16 this mutant has many swollen hyphae but unlike col-16, the col-17 mutant does not form more dichotomous branches than the wild type. smco-1 (Fig.9). The smco-1 mutant showed a flat, open growth. The colony was initially restricted but it escaped restricted growth. The main hyphae formed many branches very close to each other at the tips. Dichotomous branching was more frequent compared to wild type. The mutant formed fewer aerial hyphae and they were uniformly distributed throughout colony. The smco-1 mutant formed a lot of conidia. smco-4 (Fig. 10). The colony of this mutant was superficially very similar to the colony of the wild type. The leading hyphae extended faster, had larger extension zones and were wider than the primary and secondary branches that they subtended. No difference was observed in the number of dichotomous branches. The smco-4 mutant formed many aerial hyphae. The mutant produced an abundance of conidia. 25 smco-5 (Fig.l 1). The smco-5 mutant was fast growing with massive production of aerial hyphae and mycelia. The colony appeared bright orange due to the large amount of conidia. No differences between this mutant and the wild type were observed with respect to hyphal elongation, branching frequency and angle of branching. This mutant formed the same number of dichotomous branches compared to the wild type. Also, growth rate of this mutant was similar to that of the wild type. However, the smco-5 mutant formed more aerial hyphae and conidia than the wild type. When grown from ascospores the smco-5 strain has colonial growth (Garnjobst and Tatum, 1967). smco-6 (Fig. 12). The smco-6 mutant exhibited very restricted growth for several days then flares appeared and the colony spread over the surface. The mutant had a very dense colony and individual hyphae were only visible at the colony's margin. The distances between branches were very short. The mutant formed more dichotomous branches than the wild type. The mutant formed short aerial hyphae and bore no conidia. The colony of this mutant has characteristic irregular outline because of flaring hyphae. smco-7 (Fig. 13). The smco-7 mutant is Neurospora ras homologue (Kana-uchi et al., 1996). The smco-7 mutant exhibited flat, open growth and the branching frequency was slightly higher than that of the wild type. Hyphae of the mutant smco- 7 did not show size hierarchy and the main hyphae were as thick as their primary and secondary branches. When grown between two sheets of cellophane branches appeared at right angles. Hyphal branches at the periphery and the inner part of the colony appeared to avoid fusion. Figure 13-1 shows a hypha growing toward the adjacent hypha (Fig. 13-2), slightly curved away but continues growing parallel to adjacent hypha. Often the apex of approaching hypha splits into two and both apices continue growing parallel to the adjacent hyphae, but in opposite directions (Fig. 13-3). Usually several of these parallel 26 hyphae grow together for a considerable distance but rarely branching. Aerial hyphae were shorter than in the wild type and conidiation was relatively sparse. Colony color was faint orange due to reduced conidia formation. sntCO-8 (Fig. 14). Hyphal morphology was not uniform throughout a colony of the smco-8 mutant. Some regions of the colony had short, thin hyphae; others had flares of thicker and more branched hyphae. Usually, several branches emerged very close to each other at the tip, making broom-like ends. Dichotomous branching was more frequent compared to the wild type. It formed a huge amount of aerial hyphae and no conidia. The main characteristics of this mutant are ragged colony shape and abundance of aerial hyphae. smco-9 (Fig. 15). The mutant smco-9 formed a very restricted colony. Hyphae grew extensively into the agar. Hyphae were thin with a bumpy appearance, similar to the hyphae of the gran mutant. The bumps were possibly aborted branches. The mutant smco-9 had a smaller branch interval. The dichotomous branching was as frequent as in the wild type. The angle of branching was not different from that of the wild type. Aerial hyphae were very short and there were no conidia. spco-4 (Fig. 16). The colony was a dense with extensive aerial growth. This aerial mycelium consisted of some very long and straight primary hyphae. Concentric rings of aerial hyphae of different size gave the colony a three-dimensional appearance. These rings were probably due to periodic variation in branching of aerial hyphae. The mutant had very fine vegetative hyphae and the diameters of the main hyphae and their branches. Bursts were evident at branch sites as well as along the entire filaments. Bursts appeared when hyphae were grown between two sheets of cellophane and on the agar surface. The dichotomous branching was more frequent than in the wild type. The mutant formed a 27 large number of hyphae beneath the agar surface. The mutant spco-4 did not make conidia. In conclusion, main features of this mutant are perfectly round colony, rings of aerial hyphae of different size, uniform hyphal diameters and extensive growth of hyphae into the medium, spco-5 (Fig. 17). The spco-5 mutant formed a restricted colony. The perimeter of the colony was denser and rose above the central part of the colony. After a few days, flares of less branched hyphae appeared. Each new flare was visible as a less dense section of the colony. Because of this, the fully-grown colony appeared in distinct, terraced layers. The mutant had fine hyphae and dichotomous branching was more frequent than in the wild type. There were no leading hyphae and there was no difference in the diameters of the main hyphae and their branches. The spco-5 strain formed a large amount of mycelium beneath the agar surface. spco-6 (Fig. 18). The spco-6 mutant formed hyphae with wavy walls, and the angle of branching was more acute than in the wild type. The mutant had a high frequency of branching and many hyphae had broom-like ends. The dichotomous branching was more frequent than in the wild type. The mutant spco-6 showed extensive growth of mycelium in the medium. The spco-6 strain formed a ring of very dense and long aerial hyphae and conidiated profusely. Conidia appeared a day earlier than in the wild type. Main features of this mutant are a curly hyphae and broom-like hyphal ends. spco-7 (Fig. 19).The spco-7 mutant had very restricted growth. The colony grew as a mound; the center was raised above the perimeter, giving the appearance of concentric circles. When grown on crossing medium, spco-7 changed the color of the medium to a dirty yellow. Hyphae were very thin and the angle of branching was more obtuse than that of the wild type. No difference was observed in the number of dichotomous 28 branches. Many hyphae showed a tendency to burst. Bursts appeared at any point along the hyphal length and very often they occured at the branch sites. What made this mutant different from other mutants with regards to bursting tendency was that tips of this mutant usually did not burst. The mutant made few conidia. spco-9 (Fig.20). The spco-9 mutant formed more dichotomous branches than the wild type. Also trichotomous branching was often seen. The angle of branching was more acute than that of the wild type. Mutant spco-9 made few aerial hyphae. Vegetative hyphae had a bumpy appearance similar to the hyphae of gran except that they were thinner. This mutant formed more dichotomous branches than the wild type. The mutant exhibited very slow growth for the first few days after inoculation then flares of less branched hyphae appeared and covered the agar surface. The colony of spco-9 had several rings. The central layer was whitish, followed by the dark orange circle and each successive new ring was thinner and of a lighter yellow shade. spco-10 (Fig.21). Number, size and position of aerial hyphae made the spco-10 colony easy to distinguish from colonies of other morphological mutants. The spco-10 mutant formed large masses of aerial hyphae and conidia, many more than in the wild type. Aerial hyphae were concentrated more in the central part of the colony. In contrast, the wild type forms more aerial hyphae at the perimeter. Also aerial hyphae of spco-10 were longer compared to those of the wild type. Diameters of the main hyphae and their primary and secondary branches were the same. The angle of branching was more acute than that of the wild type. Dichotomous branches appeared more often than in the wild type. Mutant spco-10 formed large number of hyphae in the medium. In conclusion, main characteristics of this mutant are uniform hyphal diameters, characteristic position, size and amount of aerial hyphae. 29 spco-11 (Fig.22). The spco-11 mutant initially grew into a mound and subsequently formed a flat, round, slow growing colony with very fine aerial hyphae. After several days the colony escaped from its restricted growth and grew in a flat manner to cover the agar surface. The colony had a grainy texture with an orange center surrounded by a circle of dark gray spots and a whitish periphery. The mutant spco-11 formed an abundance of hyphae below the agar surface. Dichotomous branching was more common than in any other morphological mutant examined. Also, multiple branches at the apex occurred very often in this mutant. Bursts of the hyphae often occurred either at the tips or at the branch sites. The main features of this mutant are a round, flat colony, extensive growth of hyphae into the medium and many dichotomous branches. spco-12 (Fig.23). The colony of this mutant was dense and very restricted. Hyphae were very short, with spherical elongations between septa and many hyphal tips were swollen. The frequency of dichotomous branching was much higher than in the wild type. The mutant did not make conidia. frost (fr) (Fig.24). The frost gene is a homologue of the yeast cdcl both genes are involved in C a 2 + and M n 2 + homeostasis (Sone and Griffiths, 1999). The frost mutant exhibited spreading colonial growth. It formed a flat star-like colony on the surface of the medium. The colony color ranged from the pale yellow to faint orange. The mutant showed little growth of hyphae beneath the agar surface. The frost mutant had delicate hyphae with many aborted branches. Distances between branches were very short. Leading hyphae escaped from the dense colony periphery; for a while they extended like wild type, then multiple branches appeared at their tips following by growth arrest. The colony shape was irregular and deeply lobed with many uncovered patches of agar inside the colony. This mutant formed more dichotomous branches than the wild type. The frost 30 mutant formed a small number of very long aerial hyphae. Aerial hyphae were less branched and distances between branches were longer than in vegetative hyphae. The frost mutant did not make conidia. gran (Fig.25). The gran mutant formed a flat colony with grainy texture and with a few short aerial hyphae at the perimeter. Growth rate was almost as high as that of the wild type. Hyphae had a knobbed appearance, apparently due to abortion of branches. The gran mutant formed as many dichotomous branches as the wild type. The gran mutant produced a few conidia only when grown in the light. Main features of this mutant are many aborted branches and grainy texture of the colony. spray (sp) (Fig.25). The colony of the spray mutant had a irregular, lobbed shape with very long aerial hyphae. Hyphae were similar to those offrost but were thinner. The mutant spray showed a high frequency of branching. Dichotomous branching was very frequent. The mutant formed numerous of aerial hyphae. Conidia were present only when exposed to light. The product of spray gene is located in an organellar membrane, regulating the distribution of C a 2 + via calcineurin (Bok et al., 2001). peak {pk) (Fig.27). The peak mutant had the slowest growth of all examined morphological mutants. It formed very compact, lumpy, thick, light orange colonies. Colony density was not uniform throughout colony; some parts of the colony had more branched hyphae than other regions of the same colony. Usually several leading hyphae escaped from the main colony, forming flares. This mutant formed a few short aerial hyphae. Vegetative hyphae were also short and distended. Swollen hyphal tips were widespread throughout the colony and especially extensive swelling occured at tips of flaring hyphae. Apical, dichotomous, branching dominated over the lateral branching. 31 Sometimes several branches emerged very close to each other at the same tip forming star-like ends. The mutant formed conidia only when grown in the light. In conclusion, main features of this mutant are large number of dichotomous branches and star-like ends. snowflake (sn) (Fig.28). The colony had a dense, dark orange center and a less dense, whitish perimeter. Hyphae were somewhat similar to those of the frost mutant, but were thinner. This mutant formed many aborted branches. The angle of branching was less than that of the wild type. Dichotomous branching was much more frequent than in the wild type. The mutant formed a lot of aerial hyphae and conidia. Aerial hyphae were absent from the center and distributed throughout the perimeter. Main features of this mutant are many aborted branches and characteristic snowflake appearance of the colony. COt-1 (Fig.29). The cot-1 gene encodes the member of the protein kinase family (Yarden et al., 1992). The colony and hyphal morphologies of the cot-1 mutant grown at 25 °C did not differ from that of the wild type. No difference was observed in the number of dichotomous branches. Shifted to a non-permissive temperature, 37 °C, the cot-1 mutant first ceased growth then after few hours continued colonial growth. Hyphal growth under non-permissive conditions was characterized by profuse branching and bulbous hyphal tips. The cot-1 mutant formed as many aerial hyphae and conidia as the wild type at 25 °C. However, it was aconidial at 35 °C, and formed a few aberrant aerial hyphae. ipa (Fig.30). The colony was flat and less dense than the wild type. Because of that, it was possible to trace some hyphae from the point of inoculation right to the edge of the colony. The ipa mutant exhibited almost only surface growth, with less extensive growth of hyphae into the medium than the wild type and any other examined morphological mutant. The mutant ipa had many aborted branches. Dichotomous branching was rarely 32 observed. The ipa mutant did not show difference in branch interval of primary branches compared to the wild type but it did form fewer secondary branches. Tertiary branches were never observed. Aerial hyphae had a normal appearance but were fewer in number compared to the wild type, and they made less conidia. gna-1 (Fig. 31). The gna-1 encodes the a subunit of the heterotrimeric G protein (Turner and Borkovich, 1993). The mutant gna-1 had slightly slower growth compared to the wild type. The mutant had a smaller branching interval than the wild type. The mutant formed as many dichotomous branches as the wild type. Also, aerial hyphae were less abundant and they were longer than that of the wild type. The mutant gna-1 formed fewer conidia than the wild type. curly (cy) (Fig.32) The hyphae of the cy mutant were curved with many aborted branches. The cy mutant formed as many dichotomous branches as the wild type. The mutant formed few aerial hyphae and some conidia. Crooked hyphae are main characteristic of this mutant. scruffy (scr) (Fig. 33).The scr mutant hyphae had lower branch interval than in the wild type, also many aborted branches. The angle of branching was almost 90. Dichotomous branching was very frequent. The scruffy mutant formed few aerial hyphae and conidia. crisp-1 (cr-1) (Fig.34). The crisp-1 encodes adenylil cyclase (Kore-Eda et al., 1991). The cr-1 mutant formed a small, flat colony with very short aerial hyphae. Hyhae were fine and branches appeared at right angles. Dichotomous branching was not observed. Conidiation was profuse. mcb (Fig. 3 5). The mcb gene encodes the regulatory subunit of cAMP dependent protein kinase A (Bruno et al., 1996). Grown at 25 °C the mcb mutant formed fuzzy, whitish 33 colonies. Hyphae were thick with blunt tips. There was no difference in size between the main hyphae and their primary and secondary branches. The angle of branching was larger than in the wild type. The mcb mutant produced many short aerial hyphae and some conidia. coil (Fig. 36). Hyphal diameters did not differ from the wild type. The mutant also showed a size difference between main hyphae and their branches. The angle of branching was less than that of the wild type. Dichotomous branching was as frequent as in the wild type. The mutant produced fewer aerial hyphae and conida compared to the wild type. delicate (del) (Fig. 3 7) The colony of the del mutant had whitish center and dense, orange perimeter. Hyphae of the del mutant had small branch interval branches and the branches were thinner than the wild type pheripheral hyphae were slightly swollen. No difference in frequency of dichotomous branching was observed compared to the wild type. Avoidance reactions were observed as in smco-7. The mutant produced few very fine aerial hyphae and some conidia. Some of the features of the morphological mutants are summarized in Table 2. 34 Table 2. Summary of quantitative and qualitative characterization of morphological mutant phenotypes on Vogel s medium at 25 °C. (-) indicates no difference from the wild type control; (+) indicates moderate increase than in the wild type control; (++) indicates a much higher rate than in the wild type control. mutant growth rate dichotomous conidia number of colony mm/h s.e. branching branches per unit lenght density wild type 2.11 -0.18 - yes - -col-1 0.11 -0.02 - no ++ ++ col-4 0.61 -0.14 - yes + + col-8 0.12 -0.04 - no ++ ++ col-12 0.12 -0.03 + yes ++ ++ col-15 0.09 -0.01 ++ yes ++ ++ col-16 0.55 -0.11 ++ yes + +. col-17 0.91 - 0.22 - yes. + + smco-1 0.71 -0.14 + yes - -smco-4 0.94 -0.09 - yes + + smco-5 1.89 -0.08 - yes - -smco-6 0.28 -0.08 + no ++ ++ smco-7 0.82 -0.11 - yes - -smco-8 0.88 -0.16 + no + + smco-9 0.12 -0.03 - no ++ ++ spco-4 0.58 -0.24 + no + ++ spco-5 0.24 -0.02 + no ++ ++ spco-6 0.23 -0.06 + yes + + spco-7 0.32 -0.03 - yes ++ ++ spco-9 0.68 -0.12 + no - + spco-10 0.72 -0.02 + yes + -spco-11 0.51 -0.14 ++ no + + spco-12 0.08 -0.04 + no ++ ++ fr 0.82 -0.08 ++ no ++ + sp 0.71 -0.12 ++ yes ++ ++ sn 0.44 -0.13 ++ yes ++ + gran 1.46 -0.24 - yes + + cr-1 0.33 -0.04 - yes + -H-gna-1 1.33 -1.18 - yes + + cot-1 1.96 -0.23 - yes - -mcb 0.69 -0.12 - yes - + coil 1.34 -0.21 - yes - -cy 1.03 -0.52 - yes - -del 0.37 -0.11 - yes + + ipa 1.33 -0.05 - yes - -35 scr 0.97 -0.18 + yes + -pk 0.08 -0.01 ++ yes ++ ++ 2. 4. Discussion 2.4.1. Vegetative Hyphae The wild type hyphae of N. crassa (Fig. 1) exhibit a size hierarchy; the main hyphae have larger diameters than their branches. Also, N. crassa utilizes mainly lateral branching; dichotomous, apical branching occurs rarely in the wild type. In contrast, many of the morphological mutants examined did not show any difference in the size of main hyphae and branches and apical, dichotomous branching was more frequent compared to that of the wild type. Overall, the majority of the morphological mutants exhibited decreased growth rates, increased colony density and shorter branch interval relative to the wild type strain. This qualitative characterization is summarized in Table 1. The mutants col-1 (Fig.2), col-15 (Fig.6), smco-1 (Fig.9), smco-6 (Fig. 12), smco-9 (Fig. 15), spco-4 (Fig. 16), spco-7 (Fig. 19), spco-9 (Fig.20), spray (Fig.26), mcb (Fig.35), and cr-1 (Fig.34), and did not show a hyphal size hierarchy; diameters of the main hyphae and their branches did not differ appreciably. Also many branches of the mutants col-17 (Fig.8), col-16 (Fig.7), spco-5 (Fig. 17) and spco-9 (Fig.20) had larger diameters than the main hyphae, due to swelling of hyphal subapical and apical compartments. Morphological mutants col-12 (Fig.5), col-15 (Fig.6), smco-1 (Fig.9), smco-6 (Fig. 12), smco-8 (Fig. 14), spco-4 (Fig. 16), spco-5 (Fig. 17), spco-6 (Fig. 18), spco-9 (Fig.20) and spco-10 (Fig.21) exhibited a moderate increase in the frequency of dichotomous branching compared to the wild type strain (Table 1). Several mutants, col-15 (Fig.6), col-16 (Fig.7), spco-11 (Fig.22),yr (Fig.24), sp (Fig.26), sn (Fig.28) and pk (Fig.27), 36 exhibited a much higher frequency of dichotomous branching relative to the wild type. The highest frequency of dichotomous branching was noted in the spco-11 mutant (Fig.22). This mutant formed many dichotomous branches; actually, the dichotomous branching was as frequent as the lateral branching. Also, some strains did not show a consistent pattern of growth. Mutants pk (Fig.27), col-15 (Fig.6), col-16 (Fig.7) and col-8 (Fig.4) to a lesser extent, showed regional variation with respect to growth pattern. They all formed very dense, hyper-branched colonies but some parts of their colonies had less branched hyphae and those parts appeared less dense. Hyphal morphologies of mutants col-16 (Fig. 7) and col-17 (Fig.8) were grossly altered and very similar to the reported hyphal morphology of the Neurospora chitin synthase mutant chs-1 (Yarden and Yanofsky, 1991). Like the hyphae of chs-1, the hyphae of these two mutants have bulbous, swollen subapical compartments and tips. So far two chitin synthase genes have been cloned chs-1 (Yarden and Yanofsky, 1991) and csh-2 (Din and Yarden, 1994); the latter one has less altered morphology. Results from studies of Yarden and Yanofsky indicate that, like yeast, Neurospora has more than two chitin synthase genes. In addition, the double mutant chs-1;chs-2 has not been studied. So, it is possible that other chitin synthases exist in Neurospora. The hyphal appearance and bursting tendency of the hyphal tips of col-16 (Fig.7) and col-17 (Fig.8) mutants might indicate that they have altered cell walls. 2. 4.2. Aerial Hyphae and Conidiogenesis In addition to altered morphology of vegetative hyphae, aerial hyphae were also altered in all but the cot-1 strain. Either the mutants formed fewer aerial hyphae or aerial hyphae with aberrant morphology or distribution. One third of the examined strains were aconidial. Also the mutants that did form conidia usually formed less than the wild type 37 control, or conidiation showed some other abnormalities. Grown at 25 °C on Vogel's minimal media, mutants col-1 (Fig.2), col-8 (Fig.4), smco-6 (Fig. 12), smco-8 (Fig. 14), smco-9 (Fig. 15), spco-4 (Fig. 16), spco-5 (Fig. 17), spco-9 (Fig.20), spco-11 (Fig.22), spco-12 (Fig.23) and frost (Fig.24) did not produce any conidia. Three other mutant strains gran, pk and spray produced some conidia only when they were grown in the presence of light. The mutants col-15 (Fig.6) and col-12 (Fig.5) formed conidia only in test tubes and conidia were never observed in plates. In contrast, two other morphological mutants smco-5 (Fig. 11) and spco-10 (Fig.21) made more aerial hyphae and conidia than the wild type control. The mutants col-1 (Fig.2), col-8 (Fig.4), col-12 (Fig.5), col-15 (Fig.6), smco-9 (Fig. 15), spco-4 (Fig. 16), spco-11 (Fig.22), spco-12 (Fig.23) and peak (Fig.27) had very extensive growth beneath the agar surface. On the other hand, mutants ipa (Fig.30), frost (Fig.24) and gran (Fig.25) had poor hyphal growth in the medium. 2. 4. 3. Sexual Structure Under conditions of nitrogen starvation, N. crassa differentiates multicellular female reproductive structures called protoperithecia. The mutants col-15, col-1, and col-8 formed protoperitecia in the absence of nutrient starvation. In general, morphological mutants formed less protoperithecia compared to the wild type. Notable exceptions are col-4, spco-4, smco-6, col-16 and col-17, these mutants formed abundance of protoperithecia. Protoperithecia were not observed in the mutants spco-9, spco-10, col-12 and smco-9, also, sp and pk formed few protoperithecia. 38 2.4.4. Mutants That Did Not Fit Assigned Categories Phenotypic characterization showed that some morphological mutants did not fit the assigned categories. Mutants col-4, (Fig.3) and col-17, (Fig.8) which were assigned to the true colonial category by Garnjobst and Tatum (1967), exhibited spreading colonial growth. Colonies of these mutants did not stay restricted under the optimal conditions. Like spreading colonials they slowly spread over the agar surface retaining the same growth pattern. Mutant col-16 (Fig.7) had semicolonial growth; it started growth as very restricted colony but after few days, flares of faster growing hyphae appeared and the colony spread over the agar surface. Also smco-9 (Fig. 15) did not fit the definition of the semicolonial mutants. According to definition of semicolonial mutants (Garnjobst and Tatum, 1967) the semicolonial mutants escape from the restricted growth through formation of flares of faster growing hyphae. The smco-9 mutant formed restricted colonies and colony and hyphal morphologies did not change during 7-day observation. The growth rate of this mutant was similar to the growth rates of true colonial mutants. Also semicolonial mutants smco-1, smco-4, smco-5 and smco-7 did not produce flares and all parts of colony had uniform hyphal morphology. In addition spco-5 (Fig. 17) and spco-7 (Fig. 19) had very restricted morphologies analogous to those of the true colonial mutants. Some other morphological mutants such as frost, spray, sn, gran, and mcb that are not classified as colonials, in fact grow in a spreading colonial manner. The existing classification was of only limited usefulness because many mutants showed different morphologies than expected according to their names. It is possible that these mutants were not assigned to proper categories, or they have changed since they were deposited, due to accumulation of additional mutants. 39 In the classification done by Garnjobst and Tatum (1967) true colonials, spreading colonials and semicolonials are broad categories defined around one morphological trait, mycelial growth. This study showed that morphological mutants in each of these categories showed a wide variety of branching patterns, branch intervals, colony density, growth rates and amounts of conidia. Maybe a classification based on hyphal morphology would be more useful for future studies in morphogenesis. According to their hyphal morphology 28 out of 36 examined morphological mutants could be grouped into three groups. 1. ) The group of mutants that have swollen hyphae (col-16, col-17, spco-7, pk and mcb). 2. ) The group of mutants with hyphae that do not show a hyphal size hierarchy (col-1, col-8, col-15, spco-4,, spco-11, spco-9, smco-9, smco-1, smco-6 and cr-1). 3. )The group of mutants that form many aborted branches (col-4, col-12, spco-5, spco-12, smco-4, smco-8, sp, sn, scr, ipa, del, gran and cy). 40 Figure 1. Morphology of colony and hyphae of wild type 41 Figure 5. Morphology of colony and hyphae of col-12 43 Figure 6. Morphology of colony and hyphae of col-15 Figure 12. Morphology of colony and hyphae of smco-6 Figure 13. Morphology of colony and hyphae of smco-7, I: growing hypha curves away, 2: adjacent hypha, 3: hyphal apex splits into two apices Figure 14. Morphology of colony and hyphae of smco-8 Figure 17. Morphology of colony and hyphae of spco-5 49 Figure 19. Morphology of colony and hyphae of spco-7 50 Figure 22. Morphology of colony and hyphae of spco-// Figure 25. Morphology of colony and hyphae of gran 53 Figure 26. Motphology of of colony and hyphae of spray Figure 30. Morphology of colony and hyphae of ipa Figure 33. Morphology of colony and hyphae of scruffy (scr) 57 Figure 34. Morphology of colony and hyphae of crisp-/ {cr-1) M Figure 35. Morphology of colony and hyphaae of mcb 58 Figure 37. Morphology of colony and hyphae of coil 59 C H A P T E R III 3. Epistatic Interactions among Selected Morphological Mutants of Neurospora crassa 3.1. Introduction The Fungal Genetics Stock Center has a huge collection of morphological mutants with altered hyphal tip growth and branching. A l l those morphological mutants were classified by Garnjobst and Tatum, (1967) into six broad categories. In spite of this initial work the relationships between the gene products of these mutants remain unexplored. It is known that hyphal tip growth and branching are complex processes but it is not likely that all those genes are components of different developmental pathways. It is more likely that some of them act in common developmental pathways that control hyphal tip growth and branching. In this part of the research I wanted to find out whether some of the 36 selected morphological mutants might interact, possible reflecting involvement in the same pathway. By using an epistasis criterion, these morphogenes might be grouped into common developmental pathways. Also by using epistasis criterion it is possible to deduce the order in which genes of the same pathway act. In order to use epistasis analysis we have to obtain double mutants. The best way to obtain N. crassa double mutants is through tetrad analysis, looking for non-parental ditype or tetratype tetrads that are easily recognized by the presence of the wild type ascospores. In a simple interpretation of epistasis that is usually found in textbooks the double mutant has very similar phenotype to one of the single mutant parents. According to this interpretation of epistasis i f a double mutant closely resembles one of the single mutant parents then genes are components of the same developmental pathway with the epistatic gene acting before the hypostatic one. If a double mutant has more severe phenotype 60 than both parents the genes are in different developmental pathways. It seems that this is a naiiv explanation based on very simple biosynthetic pathways or simple pathways involving phase specific genes. In reality, a minority of genes are phase specific and pleiotropic genes are more common. Also most biosynthetic and regulatory pathways are very complex with many branchpoints. Regulatory pathways in particular may have steps that have multiple inputs and outputs or additional genes that act as cofactors (Avery and Wasserman, 1992). 3.2. Material and Methods Crosses between mutant strains listed in Chapter II were made according to the standard procedure described by Davis and de Serres (1970). The mutant strain that was used as a female parent was inoculated in 18 x 150 mm tubes containing 5 ml of synthetic crossing medium (Westergaard and Mitchell, 1947) and allowed to grow at 25 °C for seven days. Usually after seven days protoperithecia developed. The excess conidia on the colony were removed by using a cotton twist and a suction device. Because reciprocal crosses were performed for all interactions, the conidia from the cotton twists were saved and used as the male parent. Conidial or mycelial suspension, 0.5 ml, of appropriate male strain was added to the tube and crosses were incubated at 25 °C until perithecia mature. 3.2.1.0ctad Analysis Once ascospores began to shoot, a small part of the mycelium containing perithecia was cut out of the medium and transferred onto a microscope slide. An 4 % agar block, about 1 cm2 in size, was placed a few millimeters above the microscope slide with the perithecia. Ascospores shoot onto the upper agar block and remained together as octads. Ascospores of each octads were isolated, one at the time, and placed into 10 x 75 mm 61 slant tubes containing 1 ml of Vogel s minimal medium. Ten or more octads were isolated from each cross. Octads were incubated at 25 °C for 7 days then they were heat shocked in a 60 °C water bath for 30 minutes. Heat shocked octads were reincubated at 25 °C until colonies were formed. Octads were categorized as parental ditypes, non-parental ditypes or tetratypes and when possible, the double mutants were isolated and compared to the parental strains. The distinct morphologies of the two parental phenotypes made isolation of the double mutant quite easy except in cases of epistasis. A double mutant showing epistasis was usually indistinguishable in the test tube from one of the single mutant parents. However, when grown on plates they often showed differences. Cumulative mutations were usually distinguishable in the test tubes. For confirmation the strains from an octad, either tetratype or non-parental ditype, were plated on Vogel s minimal medium. Subsequently, the double mutant strains and their parental mutant strains were plated on solid Vogel s medium and observed using both the dissecting and the compound microscopes for comparison of colony density, branching type and frequency, presence and absence of conidia and angle of branching relative to the parental strains. The genotype of each double mutant that showed epistasis and partial epistasis was confirmed by recovery of single mutants in a backcross to wild type. 3. 2. 2. Measurement of Growth. Measurement of double mutants and their mutant parents was done as described in Chapter II except for the group showing epistasis. Growth rates of the double mutants from that group and their parents were measured for 5 days or until colonies filled the plates. Faster growing single mutant parents were grown in large plates. The colony front was marked on five radii every day. The distances between the original point of inoculation and the markings were measured and plotted against time. 62 3.2.3. Random Ascospore Analysis Random ascospores were isolated according to the method of Davis and de Serres (1970). The spores were transferred from the crossing tubes onto the 1.5 X 1.5 cm blocks of 4% agar. Then ascospores were picked one at the time and placed in test tubes containing Vogel s medium. The picked spores were immediately heat shocked for 30 minutes in a 60 C water bath and incubated at 25 °C to germinate them. 3.2.4. Heterokaryon The helper strain aml ad-SB cyh-1 (Griffiths, 1982) was used in crosses between the mutant strains frost and gna-1, which are female sterile. Forced heterokaryons were constructed between the helper strain and met-3 marked frost mutant strain. The heterokaryon was used as a female mutant parent; a conidial suspension of gna-1 was added as the male parent. 3.3. Results 3.3.1. Crosses True colonial mutants were crossed with semicolonial and spreading colonial mutants, whenever compatible mating types were available. Crosses between spreading and semicolonial mutants and among mutants in each category have already been done in previous studies. In addition, several other morphological mutants have been used because of their interesting phenotypes or because they were defective in homologues of genes that are components of signaling pathways in other organisms. These mutants were crossed to true colonial, semicolonial and spreading colonial mutants, whenever two mutants were distinguishable and compatible mating types were available. Table 13 lists all attempted crosses. 63 A total of 381 reciprocal crosses were attempted between these single mutant strains and 102 double mutants were recovered from those crosses. There were 38 double mutants that closely resembled one of the single mutant parents in hyphal morphology. However, this high degree of similarity in hyphal morphology was not necessarily accompanied by similarity in colony morphology. In addition 16 out of 41 double mutants that showed an additive phenotype did not have more severe phenotypes than both parents. Also 23 double mutants did not show similarity to any of the parents and some of them resembled some other morphological mutants that were not parental strains. Acording to their phenotypes all double mutants obtained in this study were divided in 5 groups. 1.) The group showing full epistasis included the double mutants that closely resembled one of the mutant parents in hyphal morphology and colony shape but not necessarily size. 2. ) The group showing partial epistasis included double mutants that closely resembled one of the single mutant parents in hyphal morphology but not in colony shape and size. 3. ) The group showing coepistasis included double mutants that showed features of both parents. 4. ) The group showing cumulative effects of both mutations included double mutants that had more extreme phenotypes than either of the parents 5. ) The group called novel included double mutants that did not show similarity to any of the parents in hyphal and colony morphiologies as well as double mutants that resembled some other morphological mutant, which was not of a parental strain. 64 3.3.2. Phenotypic Characterization of Double Mutants 3.3.2.1. Group of Double Mutants Showing Full Epistasis A l l double mutants from this group showed high similarity in hyphal and colony morphologies to one of the single mutant parents. The phenotypes of the double mutant and the single mutant were indistinguishable in test tubes. Grown on plates the double mutant showed high similarity to the single mutant parent in hyphal morphology. Usually it was not possible to distinguish hyphae of the double mutant and hyphae of one of the parents. According to epistasis criterion those genes are components of the same developmental pathway involvd in hyphal tip growth and branching. The colony of the double mutant was similar in shape to the colony of one parent but it was usually somewhat smaller in size. Growth rates of double mutants and their single mutant parents are summarized in Table 10. Some of the features of double mutants showing epistasis are summarized in Table 3. 65 Table 3. Summary of qualitative and quantitative characterization of double mutants showing epistasis. (+) indicates conidia, (*) conidia formed only in test tube, bold letters show epistatic mutation double mutant growth rate mm/h conidia descriptive notes spco-11,-col-8 (Fig. 39) 0.13 -restricted colony growth -growth rate similar to col-8 -extensive growth of hyphae into the medium smco-8;pk (Fig.40) 0.09 -hyphae resembled pk -many dichotomous branches -star-like ends -hyphal tips often burst -frequent flares of swollen hyphae -restricted colony similar to pk, -growth rate similar to pk -three-D appearance of colony spco-1 l;col-l5 (Fig.41) 0.11 + * -hyphae similar to col-15, -growth rate similar to col-15 -dichotomous branching -flares of thick hyphae -restricted colony growth, -extensive growth in medium smco-5 fr (Fig.42) 0.78 -hyphae resemble frost -growth rate similar to frost -dichotomous branching -many aborted branches -colony similar to frost -larger number of aerial hyphae than in frost col-16;fr (Fig.43) 0.29 + -hyphae very similar to col-16, - swollen hyphal tips -hyphal tips often burst -frequent dichotomous branching -balls of conidia spco-4 ;col-l 5 (Fig. 83) 0.12 + * -hyphae resemble those of col-15 -some dichotomous branches -extensive growth of hyphae in the medium -very small colony -growth rate similar to col-15 col-15;smco-7 (Fig.84) 0.11 + * -hyphal morphology similar to col-15 -some flaring hyphae -frequent dichotomous branching -very restricted colony similar to 66 col-15 -growth rate similar to col-15 -dark orange colony center col-4;fr (Fig.85) 0.32 -hyphae similar to frost -many aborted branches -some dichotomous branches -ragged colony shape -few aerial hyphae col-4;spco-12 (Fig.86) 0.07 - -similar growth rate to spco-12 -hyphae resemble spco-12 smco-6;col-8 (Fig.87) 0.12 - -similar growth rate to col-8 -hyphal resemble col-8 smco-7;cr-l (Fig.88) 0.28 -hyphae resembele cr-1 -growth rate similar to cr-1 -colony shape similar to cr-1 -very fine, short aerial hyphae -right angle branching col-15 gna-1 (Fig.89) 0.08 -hyphae very similar to col-15 -flares of faster growing hyphae -colony morphology very similar to col-15 -growth rate similar to col-15 mcb;cr-l (Fig.90) 0.52 + -hyphae similar to cr-1 -colony resembles cr-1 -very fine, short aerial hyphae col-16 gna-1 (Fig. 91) 0.52 + -hyphae resemble col-16, -swollen hyphal tips - hyphal tips often burst -frequent dichotomous branching -flares of faster growing hyphae -colony resembles col-16 -growth rate similar to col-16 -balls of conidia ipafr (Fig.92) 0.87 -hyphae very similar to frost, -dichotomous branching -many aborted branches -colony similar to frost but has a less ragged colony shape -growth rate similar to frost -few long aerial hyphae smco-7 mcb (Fig.93) 0.78 -hyphae resemble smco-7 -right angle branching - hyphae avoid to fuse -colony show similarity to both parents -dark orange colony - fuzzy appearance 67 coil;fr (Fig.94) 0.69 - -hyphae similar to frost, -dichotomous branching - many aborted branches 3.3.2.2. Group of Double Mutants Showing Partial Epistasis In this group it was not possible to distinguish the hyphal morphologies of the double mutant and one of the single mutant parents, but the colony of the double mutant differed in size and shape from the colony of the epistatic parent. The double mutant always formed a smaller colony compared to the epistatic parent. Also, a notable difference occurred in aerial hyphae formation; usually the double mutant formed fewer aerial hyphae, which were in some cases shorter and differently positioned compared to the epistatic parent. Those genes might be the components of the same developmental pathway involved in vegetative hyphe growth. Some of characteristics of double mutants showing partial epistasis are summarized in Table 4. 68 Table 4. Summary of qualitative and quantitative characterization of double mutants showing partial epistasis. (+) conidial, (-) acondial double mutant growth rate mm/h conidia descriptive notes smco-7; gna-1 (Fig.44) 0.61 + -hyphae resemble smco-7 -flat colony growth -very short aerial hyphae -right angle branching -hyphae do not fuse -uplifted center of colony spco-11 ;spco-10 (Fig.45) 0.47 + -colony resemble spco-11 -extensive growth into the medium -hyphae very similar to spco-10 -uniform diameters of main hyphae and branches -some dichotomous branches smco-8;Jr (Fig.46) 0.31 -hyphae indistinguishable from the hyphae of frost -ragged colony shape -more restricted colony than frost scr;fr (Fig.47) 0.45 -hyphae resemble frost -dichotomous branching - many aborted branches -colony shape similar to frost but colony is more restricted -slower growth rate than in any of the parents - few long aerial hyphae col-4 ;col-17 (Fig.48) 0.57 + -hyphae very similar to col-4, -different diameters of main hypahae and branches -many aborted branches -growth rate similar to col-4 col-16;spco-6 (Fig.95) 0.21 + -hyphae resemble spco-6 -growth rate similar to spco-6 spco-4;col-l 7 (Fig.96) 0.37 -hyphae resemble spco-4 -rings of aerial hyphae of different size -slower growth rate than in any of the parents -colony shape similar to spco-4 col-4;gran (Fig.97) 0.39 -hyphae similar to col-4 -many aborted branches -colony similar to col-4 but more restricted 69 -slower growth rate than in both parents -different color of colony than col-4 probably due to absence of conidia spco-4;fr (Fig.98) 0.49 -hyphae resemble frost -growth rate more similar to spco-4 -interesting colony morphology -colony center resembles spco-4 -colony perimeter resembles frost col-4;spco-9 (Fig.99) 0.43 + -lower growth rate than in any of the parents -hyphae very similar to col-4 gran;fr (Fig. 100) 0.30 -ragged colony shape -releases some brownish pigment -hyphae very similar to frost -many aborted branches -frequent dichotomous branching mcb;fr (Fig. 101) 0.55 -hyphae resemble frost -fuzzy, whitish colony -colony was slightly denser than in mcb -growth rate similar to mcb -few long aerial hyphae spco-6;fr (Fig. 102) 0.09 -hyphae very similar to frost -colony similar to frost parent but much more restricted -growth rate much lower than in either of the parents spco-4;smco- 7 (Fig. 103) 0.39 -hyphae resemble smco-7 -right angle branching -hyphae do not fuse -intermediate colony morphology -some concentric rings similar to spco-4 spco-4 ;gna-l (Fig. 104) 0.52 -hyphae resemble spco-4 -uniform diameter of main hyphae and branches -extensive growth in the agar -colony more similar to spco-4 -growth rate similar to spco-4 -concentric rings col-4;spco-10 (Fig. 105) 0.23 + -hyphae were similar to col-4 -many aborted branches -colony shape similar to col-4, but colony was more restricted 70 -concentric rings of aerial hyphae of different size -shorter aerial hyphae than in both parents smco-7;col-17 (Fig. 106) 0.35 - -growth rate much lower than in any of parents col-15; spco-9 (Fig. 107) 0.18 -colony resembles col-15 -growth rate higher than in col-15 - hyphae resemle col-15 -aerial hyphae appeared like spikes, long,thin and almost without branches col-16;spco-10 (Fig. 108) 0.60 + -restricted colony -hyphae similar to col-16 -swollen hyphal tips -frequent hyphal bursts col-4;gna-1 (Fig. 109) 0.58 + -more restricted colony than in the parents -hyphae resemble col-4 -many aborted branches col-16;smco-l (Fig. 110) 0.60 + -hyphae very similar to col-16 -swollen hyphal tips -dichotomous branching -fine aerial hyphae 71 3.3.2.3. Group of Double Mutants Showing Coepistasis In this group the hyphal morphology of the double mutant was a combination of both mutant phenotypes. Specific characteristics of both mutations could be easily recognized. This group included most of the double mutants involving cot-1. Most of the cot-1 double mutants phenotypically resembled the other mutant parent at 25 °C, because the cot-1 mutant at 25 °C is essentially a wild type. However, the double mutants changed their branching patterns at non-permissive temperature (above 32 C) in the presence of cot-1. Those genes probably act in parallel pathways. The main characteristics of double mutants showing coepistasis are summarized in Table 5. 72 Table 5. Summary of quantitative and qualitative characterization of double mutants showing coepistasis, (+) conidial, (-) aconidial double mutant growth rate mm/h conidia descriptive notes smco-7;gran (Fig.49) 1.62 -more restricted growth than in any of the parents -size of hyphae and right angle of branching similar to smco-7 -many aborted branches similar to gran -hyphae avoid fusion col-16;mcb (Fig.50) 0.57 + -colony appearance more similar to mcb -dichotomous branches -large amount of conidia -swollen hyphal tips col-16;col-4 (Fig.51) 0.51 -swollen hyphal tips -many aborted branches -colony more restricted than in any of the parents -frequent dichotomous branching -no aerial hyphae spco-6;gran (Fig.52) -main hyphae are curly and similar to spco-6 -many aborted branches similar to gran -dark yellow colony with some brown spots -scarce aerial hyphae smco-6;fr (Fig.53) 0.18 --most of colony had hyphae similar to smco-6 -flares offrost —like hyphae -colony more restricted than either of the parents col-16;smc5 (Fig . I l l ) 0.32 -restricted colony -thick and swollen main hyphae -many burst tips -numerous dichotomous branches spco-1 l;gran (Fig.112) 0.78 -main hyphae resemble spco-11 -many aborted branches similar to gran -unlike gran, which had only initiation of tertiary branches the double formed tertiary branches -frequent dichotomous branching -dark yellow colony 73 cot-1 ;gran (Fig. 113) 1.51 -hyphae and colony resemble gran when grown at permissive temperature -when exposed to light at 25 °C the double mutant form some conidia -restricted growth at non-permissive temperature smco-7; col-4 (Fig. 114) 0.39 - -restricted colony growth -few short aerial hyphae spco-4;gran (Fig. 115) 0.52 -colony similar to spco-4 -concentric rings of aerial hyphae of different size -extensive growth in medium growth rate similar to spco-4 smco-7;cot-l (Fig. 116) 1.15 -hyphae and colony resemble smco-7 when grown at permissive temperature -fine, short aerial hyphae -restricted growth at non-permissive temperature col-4 cot-1 (Fig. 117) 0.69 + -when grown at permissive temperature the double mutant hyphae and colony resemble col-4 -whitish colony -grown at non-permissive temperature hyphae and colony resemble cot-1 col-15; spco-7 (Fig.118) 0.18 -hyphae of the main trunk of the colony resemble the hyphae of col-15 -flares resemble spco-7 -colony more similar to col-15 spco-4;cot-l (Fig.l 19) 0.56 -hyphae and colony of the double mutant resemble the spco-4 mutant at permissive temperature -concentric rings -very restricted growth at non-permissive temperature smco-5 ;col-l 7 (Fig. 120) 0.34 + -very thick, swollen hyphae -frequent hyphal bursts -restricted colony growth -rounded hyphal tips -frequent dichotomous branching -lower branch interval than in col-17 74 cot-l;col-16 0.32 - -hyphae and colony very similar to (Fig.121) col-16 at permissive temperature -swollen hyphal tips -hyphal tips often burst -many dichotomous branches -regional difference in colony density -restricted growth at non-permissive temperature 3.3.2.4. Group of Double Mutants Called Novel Double mutants from this group usually did not show similarity to either of the parents. Differences in colony and hyphal morphologies were quite obvious. Interestingly some of those mutants showed high similarity to some other morphological mutant that was not a parental strain. The similarity was either in hyphal or colony morphology or both. Probably that was due to some degree of interconnection of different pathways that regulate hyphal tip growth and branching. Those genes could be components of different developmental pathways involved in hyphal tip growth and branching. The features of double mutants showing a novel phenotype are summarized in Table 6. 75 Table 6. Summary of quantitative and qualitative characterization of double mutant showing a novel phenotype, (+) conidial, (-) acondial double mutant growth conidia descriptive notes rate mm/h smco-8;col-15 (Fig.54) 0.41 -colony less restricted than colony of col-15 -higher branch interval -frequent dichotomous branching spco-11; gna-1 (Fig.55) 0.66 -colony similar to spco-11 -faster growth than spco-11 -fine, short aerial hyphae gran;spco-12 (Fig.56) 0.23 -intricate branching pattern -dense colony -three-D colony appearance col-16; gran (Fig-57) 0.06 -hyphae similar to spray -bumpy appearance of hyphae -frequent dichotomous branching spco-4;col-l (Fig.58) 0.31 -less restricted colony than in col-1 -improved hyphal morphology -different diameters of main hyphae and branches -angle of branching similar to that of wild type -blunt and slightly swollen hyphal tips -hyphae are similar to del gran;col-l (Fig.122) 0.60 -most of hyphae resemble col-1 -very dense colony and only tips of individual hyphae were visible -dark orange colony without aerial hyphae -frost-like flaring hyphae spco-4;pk (Fig. 123) 0.21 -slower growth, some vertical build-up -swollen hyphal tips -dichotomous branching was more frequent than the lateral one col-16; spco-5 (Fig. 124) 0.38 -colony grows as a mound -many swollen hyphae -frequent bursts of hyphal tips 76 spco-6; smco-9 (Fig. 125) 0.10 + -very restricted colony growth -uniform size of hyphae smco-9; col-16 (Fig. 126) 0.19 -thin, curly hyphae -frequent dichotomous branching cr-l;cot-l (Fig.127) 0.31 + -numerous aerial hyphae -very long aerial hyphae, which are radially distributed col-15 col-16 (Fig.128) 0.08 - -colony similar to col-15 -dark orange colony center smco-6; col-16 (Fig. 129) 0.12 -three-D colony appearance -bumpy appearance of hyphae -few aerial hyphae smco-7; col-16 (Fig.130) 0.31 -small, dense colony -only swollen tips of individual hyphaewere visible -few aerial hyphae smco-8;spco-4 (Fig.131) 0.13 -very restricted colony -hyphae similar to frost -bumpy appearance of hyphae probably due to many aborted branches gran;cy (Fig.132) 0.98 - -curly hyphae -cottony aerial hyphae spco-11; col-1 (Fig.133) 0.09 -hyphae of main trunk of the colony have low branch interval -many flaring hyphae -no dichotomous branches spco-4;mcb (Fig.134) 0.54 -round colony -many concentric rings -very short aerial hyphae mcb;col-l 7 (Fig.135) 0.03 -very restricted colony growth -many swollen hyphae -hyphal.tips often burst smco-6;col-l 7 (Fig. 136) 0.10 -compact colony -thin hyphae -larger branch interval than in smco-6 smco-6; col-15 (Fig.137) 0.07 -numerous dichotomous branches -flaring hyphae cot-l;gna-l (Fig.138) 1.49 + -numerous aerial hyphae -right angle branching mcb;cot-l (Fig.139) 0.62 -when grown at permissive temperature hyphae are very similar to mcb 11 -colony appearance similar to mcb -restricted growth at non-permissive temperature 3.3.2.5.Group of Double Mutants Showing Cumulative Effects of Both Mutations The group showing cumulative interaction was the most abundant one. The phenotypically visible double dose of both mutations indicates that these genes operate in separate pathways involved in hyphal tip growth and branching and do not undergo any direct interactions. According to the epistasis criterion the genes are in parallel pathways. These double mutants usually grew vertically into mounds, rather than spread horizontally on the agar surface; usually colony of the double mutant was more restricted than the colony of more restricted parent. Also, the hyphal morphology of the double mutant was more altered than either of the parents. Hyphae often showed isotropic growth that resulted in some spherical compartments between septa. Sometimes hyphae of the double mutants were so tiny that they formed very dense colonies between two sheets of cellophane, making it impossible to deduce the branching pattern. In most cases the double mutant was aconidial. Table 7 summarized the features of the double mutants showing cumulative effect of both mutations. 78 Table 7. Summary of quantitative and qualitative characterization of the double mutants showing cumulative effect of 30th mutations. double mutant growth rate mm/h conidia descriptive notes smco-7;fr (Fig.59) 0.01 -tiny colony -colony growth was noted only first 24 hours -few tiny flares of very fine hyphae -no aerial hyphae cot-1 ;fr (Fig.60) 0.01 -tiny colony -one of the slowest growing double mutant - bead-like hyphae -hyphal morphology grossly altered -hyphae lost polar growth -some hyphae escape from the isotropic growth but soon they restart hyperbranching and isotropic growth -few aerial hyphae with similar morphology to cot-1 -no difference in growth between permissive and non-permissive temperature smco-8 col-4 (Fig.61) 0.13 -very severe phenotype -many hyphae utilized isotropic growth -no aerial hyphae smco-8; smco-7 (Fig.62) 0.16 + -small, dense colony -flares of some larger hyphae -many bursts occurred at the branch sites smco-8;cot-l (Fig.63) 0.21 + -restricted growth at both, permissive and non-permissive temperature -no aerial hyphae spco-4; col-4 (Fig. 140) 0.31 -concentric rings -bumpy hyphae -many aborted branches col-4;spco-ll (Fig. 141) 0.20 -tiny colony -more restricted growth than in any of the parents -bumpy, hyperbranched hyphae -no dichotomous branching col-8;col-17 (Fig. 142) 0.08 - -dense, lumpy colony -colony growth more restricted 79 than either of the parents -flares of thick hyphae with some swollen tips -lateral branching smco-8;smco-6 (Fig. 143) 0.14 -hyphal morphology similar to smco-6 mutant -restricted colony growth -no flares smco-8;spco-5 (Fig. 144) 0,09 -shape of colony similar to spco-5 but colony was more restricted -few flaring hyphae -frequent dichotomous branching -very short branch interval col-16;pk (Fig. 145) 0.06 -very compact colony -only tips of individual hyphae were visible -some flaring hyphae with swollen, round tips -many dichotomous branches snfr (Fig. 146) 0.12 -colony growth more restricted than either of the parents -lower branch interval than in parents -colony has grainy texture -bumpy hyphae probably due to aborted branches cot-1 ;col-17 (Fig. 147) 0.06 -dense, restricted colony -few flaring hyphae -no difference in hyphal and colony morphology when grown at non-permissive temperature -no aerial hyphae spco-12 fr (Fig. 148) 0.05 - -restricted colony growth -no aerial hyphae col-4;spco-6 (Fig. 149) 0.34 -low branch interval -no aerial hyphae -more restricted growth than in any of the parents smco-6; col-4 (Fig. 150) 0.62 - -small, compact colony col-4; spco-5 (Fig. 151) 0.14 + -colony growth more restricted than in any of the parents -some dichotomous branches col-15;fr (Fig. 152) 0.06 -restricted colony than col-15 -hyphal morphology shows some similarity to frost, especially flaring hyphae 80 sp;fr (Fig. 153) 0.29 -ragged colony shape similar to frost -slower growth than both parents -many aborted hyphae spco-11;col-17 (Fig. 154) 0.18 -bumpy appearance of hyphae -frequent dichotomous branching -lower branch interval than in any of the parents -few short aerial hyphae smco-4; col-17 (Fig. 155) 0.25 -very restricted growth -low branch interval -many swollen hyphae col-4; smco-1 (Fig. 156) 0.13 -very restricted colony -curly hyphae gran;col-l 7 (Fig. 157) 0.05 -restricted colony -distorted hyphal morphology -short branch interval -frequent dichotomous branching -flares of thick, bumpy hyphae -some aborted branches with swollen tips smco-6;col-l (Fig. 158) + -colony similar to col-1 -very short hypahe -flares of longer hyphae -forms a few conidia smco-4;col-16 (Fig.159) 0.24 - - -very,restricted colony growth -tiny hyphae 3.3.3. Grouping of Morphogenes into Common Developmental Pathways This study revealed some epistatic interactions among examined morphological mutants. In all cases of epistasis and partial epistasis the epistatic mutation was always the more restricted parent. Also, morphological mutants that had more extensive growth of hyphae 81 in the medium were always epistatic. The double mutants showing full or partial epistasis are listed in Table 8. Table 8. The double mutants showing full and partial epistasis. (*) partial epistasis, ?old letters show epistatic mutations. col-4; fr col-15; spco-9* smco-7; cr-1 smco-8;fr * spco-4;col-15 * mcb;cr-l gran; fr * col-15; smco- 7 spco-4;smco-7 * scr; fr * col-4; gna-1 * smco-7; mcb ipa;fr col-15;gna-l smco-7; gna-1* coil; fr col-4;col-l 7 * col-16 ;fr mcb; fr * col-4;gran * col-16; gna-1 spco-4;fr * col-4;spco-10 * col-16; spco-10* spco-6; fr * col-4;spco-9 * col-16; smco-1* smco-5; fr smco-8; pk spco-11;spco-10 * spco-11; col-8 spco-4; gna-1 * col-4;spco-12 smco-6;col-8 spco-4;col-l 7* col-16;spco-6 * spco-11 ;col-15 smco-7;col-17* Some genes were involved in several cases of epistasis that gave possibility for their grouping into common developmental pathways. Four provisional pathways involved in vegetative hyphae growth are shown in Fig.38. 1. ) col-15 • smco-7-—^ spco-4 • gna-1 2. ) fr • col-4 • gran 3. ) cr-1 • smco-7 • mcb 4. ) smco-7 ^-spco-4 • col-17 Figure 38 Provisional pathways involved in vegetative hyphae growth. 3.4. Discussion 3. 4.1. Crosses About three quarters of the crosses made did not yield double mutants. To make a successful cross with morphological mutants was often difficult because of poor fertility 82 and absence of conidia. Also it was difficult to observe protoperithecia in the very dense and lumpy colonies of some of those mutants. Furthermore, mound-like colonies smothered the perithecia, trapping the ascospores ejected from the asci. Finally, the poor germination rate of ascospores often resulted in non-informative octads. A l l those crosses that did not yield double mutants have been attempted several times under slightly changed conditions; that is the female strain was grown for a longer time before the cross was made, or crosses were made in petri dishes. To make a cross on a petri dish has the advantage over a tube because of the possibility of obtaining more perithecia; however, this approach is prone to contamination. In most crosses that did not yield the double mutant, informative octads showed that double mutants did not survive. The double mutants did not survive from the following crosses smco-5 X col-1, col-1 X spco-7, col-8 X spco-9, smco-1 Xpk, spco-10 X col-17, smco-7 X col-8, col-17 X smco-8, col-4 X spco-7, col-16Xspco-7, col-16X spco-9, sp X col-4, smco-8Xgran, smco-8X mcb, sn X smco-6, sn X sp, sp X smco-8 and col-17' X fr. However, from some crosses informative octads were not obtained because of poor ascospore germination. From those crosses 50 random ascospores were picked and checked for the presence of the double mutants. In several crosses perithecia did not form either in a tube or in a petri dish; those crosses were considered infertile. A l l crosses involving col-12 produced abundance of perithecia but ascospores were not formed. In most of those crosses perithecia looked normal except that beaks were not present. Also, crosses involving sp, pk, spco-7, spco-10, spco-12, smco-1, smco-4, smco-5, smco-9and sn usually produced a few perithecia. A l l double mutants from groups showing full and partial epistasis were backcrossed to the wild type. The backcrosses resulted in a progeny phenotype ratio close to 1:1:1:1 for the double mutant, wild type and single mutant parents. The presence of the single 83 mutant parents among progeny of the backcross confirmed the identity of the double mutant. 3. 4. 2. Possible Reasons for the Complexity of Interactions Epistasis and cumulative groups of double mutants were expected. However, half of double mutants obtained in this study did not fit the traditional view of epistasis. The most interesting group of double mutants was the group showing partial epistais. Double mutants showed clear epistasis in hyphal morphology but the epistasis was absent in colony morphology or reversed. Double mutants showing epistasis and partial epistasis are listed in Table 8. Possible reasons for the results obtained in this study could be residual expression of the hypostatic gene in the absence of the epistatic one, pleiotropy or allele specific effects. In this study only one allele of each mutant was used and it is possible that other alleles would show different interactions. 3.4. 2.1. Residual Expression of Hypostatic Genes One possible reason for this complexity of interactions could be a residual expression of the hypostatic gene in the absence of the epistatic one. Most of the genes used in this study have not been studied at molecular level and it is not known if they are null-mutants or not. However, frost (Sone, 1999), gna-1 (Ivey, 1996), smco-7 (Kana-uchi, 1997) and mcb (Bruno, et al., 1996) that are null-mutants were involved in several cases of partial epistasis as the hypostatic mutation le.fr ;mcb (Fig. 101), spco-4;gna-\ (Fig. 104), cr-l;mcb (Fig.90) smco-7;mcb (Fig.93), smco-7;gna-l (Fig.44), cr-l;smco-7 (Fig.88), col-16 ;fr (Fig.42), and col-17;mcb (Fig. 107). This suggests that at least for these double mutants the residual expression was not the reason for the partial epistasis. 84 3. 4. 2. 2. Pleiotropy An other explanation for the results obtained in this study could be pleiotropy. A l l genes involved in partial epistasis are pleiotropic in the sense that mutations at these loci affect more than one phase of the Neurospora life cycle. Phenotypic characterization of these morphological mutants presented in Chapter II showed that mutations at most of these loci did not affect only vegetative hyphae growth but also aerial hyphae formation, conidiogenesis, formation of hyhae under the agar surface and sexual development. Aerial hyphae are very important for colony morphology; the amount, position and size of aerial hyphae give the characteristic appearances of each mutant strain. The amount of aerial hyphae and any abnormality during their development can have a direct impact on conidiation. It is also interesting that each of these mutants develops a different number of hyphae beneath the agar surface. It is notable that the formation of larger number of hyphae in the medium interferes with the growth rate and colony shape on the surface of the agar. Mutants that form more hyphae in the medium spread slowly on the agar surface and they form perfectly round colonies. Examples are spco-4 (Fig. 16), spco-11 (Fig.22), col-1 (Fig.2), col-8 (Fig.4). I also examined the branching patterns of vegetative and aerial hyphae of the mutants involved in partial epistasis. The branching patterns of vegetative and aerial hyphae of the wild type (Fig. 64) are the same in contrast branching patterns of vegetative and aerial hyphae of the morphological mutants involved in partial epistasis are different i.e. frost (Fig.65), spco-4 (Fig.66) and col-17 (Fig.67). Also, aerial hyphae of some double mutants resembled the hypostatic parent i.e. smco-7;gna-l (Fig.69). In general, 85 morphology of aerial hyphae, either of single or double mutants, was less severe compared to vegetative hyphae. According to results obtained in this study most of the morphological mutants used in this study are involved in conidiogenesis (see Chapter II). Even mutants that make conidia usually make fewer conidia than the wild type, (exceptions are mutants smco-5 and spco-10), or the conidia have other abnormalities. More than half of the obtained double mutants were aconidial; 26 of aconidial double mutants had both conidial parents and 38 of them had one conidial parent. Only the double mutant smco-6;col-l formed a few conidia although both mutant parents were aconidial. Conidiation patterns of double mutants and their single mutant parents are listed in Table 9. If two pleiotropic genes are components of the same developmental pathway that control vegetative hyphae formation they should have some overlapping functions. The double mutant involving those two mutations would show epistasis in hyphal morphology. However, the hypostatic gene might be involved in the formation of aerial hyphae or growth of hyphae into medium; however, the epistatic gene might not be involved in these processes. Even i f the epistatic gene is involved, it is possible that the order of gene action is not the same. In both cases the colony morphology of the double mutant would be different from the colony morphology of the epistatic mutant. 3.4. 3. Double Mutants Resembling Non-parental Morphological Mutants Several double mutants from the group showing a novel phenotype showed similarity to some morphological mutants that were not parental strains. The hyphal morphology of the double mutant spco-4;col-l (Fig.58) resembled delicate (del) (Fig.36). The hyphae of the double mutant were less altered than the hyphae of the parental mutant strains. This 86 was the only double mutant that had hyphal morphology less altered than the parents. The double mutant spco-6; gran (Fig.52) resembled spray (Fig.26) in colony and hyphal morphologies. The hyphal morphology of the double mutant smco-8;spco-4 (Fig. 131) resembled the hyphae of frost (Fig.24), especially the flaring hyphae of the double mutant, which had very similar branching pattern to frost. The frost mutation was epistatic to both smco-8 and spco-4 mutations. This might suggest interconnection of the pathways where spco-4 amd smco-8 genes act. Similarly, The frost mutation was epistatic to gran and the double mutant gran;col-l (Fig. 122) formed flaring hyphae that were very similar to those of frost (Fig.24). Approximately one third of existing Neurospora morphological mutants were used in this study and it is possible that some other double mutants from the novel group also resembled some other morphological mutants that were not included in this study. 3.4.4. Provisional Characterization of Three Pathways Involved in Hyphal Tip Growth and Branching Two mutants frost and col-15 were involved in several cases of epistasis interactions (Table 8) allowing these genes to be placed in different developmental pathways (Fig. 3 8). The presence of some cloned genes in each pathway can give some clue of their possible functions as discussed below. Pathway 1 (Fig.38) contains two known genes smco-7, which is the Neurospora ras homologue (Kana —uchi et al., 1996) and gna-thsA is gene for the a subunit of a heterotrimeric G protein (Turner and Borkovich, 1993). Homologues of both these genes are involved in pseudohyphal growth in S. cerevisiae. G proteins are components of the c A M P - P K A pathway in S. cerevisiae and other organisms. The ras proteins are involved in several different processes in various fungi. In S. cerevisiae, the Rasl and Ras2 87 proteins are essential for the regulation of cell division (Kataoka et al., 1984). In this organism the Ras2p activates both c A M P - P K A and M A P kinase pathways. Both pathways are involved in pseudohyphal growth. In the filamentous fungus A. nidulans, the roles of the ras protein homologue A-Ras is involved in the regulation of conidiophore formation (Som and Kalaparthi, 1994). In N. crassa ras homologue, smco-7 is involved in regulation of formation of aerial hyphae and conidia (Kana-uchi et al., 1997). Also, G a subunit plays different roles in various fungi. In S. cerevisiae the subunit Gpal is a negative regulator of pheromone response /mating pathway (Kurjan, 1992). In N. crassa G a subunit, gna-1, is involved in sexual development and macroconidiation, the major asexual reproduction pathway in N. crassa and (Ivey et al., 1996) . „ ; . : ". The second pathway (Fig. 3 8) contains the frost gene, which is involved in C a 2 + and M n 2 + homeostais in N. crassa (Sone and Griffiths, 1999). The frost homologue in yeast, Cdcl is also involved in C a 2 + and M n 2 + homeostasis (Loukin and Kung, 1995). The frost mutant phenotype is correctable by exogenous C a 2 + and it is sensitive to elevated M n 2 + concentrations. This study showed that col-4 is sensitive to increased concentrations of M n 2 + and unlike frost it was sensitive to elevated C a 2 + concentrations, too. In contrast, increased M n 2 + concentrations greatly improved the growth rate of the gran mutant. The third pathway (Fig. 3 8) contains three cloned genes and some of their functions are known. The Neurospora crisp-1 {cr-1) is structural gene for adenyl cyclase (Kore-Eda et al., 1991). The cr-/mutant has a low level of cAMP. In N. crassa cAMP is involved in utilization of endogenous and exogenous carbon sources, morphogenesis of vegetative hyphae, and formation of aerial hyphae and conidia (Terenzi et al., 1994; Flawia et al., 1997) . The smco-7 gene is the ras homologue and mcb is regulatory subunit of cAMP dependent P K A protein (Bruno et a l , 1996). In N. crassa the P K A pathway regulates 88 polarized deposition of wall material for hyphal growth (Bruno et al., 1996). Homologues of all three genes are components of the c A M P - P K A signaling pathway in S. cerevisiae, which is involved in pseudohyphal growth in yeast Fourth pathway contains ras homologue smco-7 and two unknown genes spco-4 and col-17. There is a possibility that the mcb and cr-1 genes are also components of the pathway 1 because mutations in spco-4 and and smco-7 genes are epistatic to mutation of mcb gene. However, the double mutants col-15 ;mcb and gna-l;mcb could not be obtained. Also the cr-1 mutation is epistatic to mcb and smco- 7 mutations. A l l attempts to obtain double mutants between cr-1 and other components of the pathway 1, gna-1 and col-15, were not successful. Also, it is possible that col-17 is component of the pathway 1 (Fig. 38). Two mutations from this pathway smco-7 and spco-4 are epistatic to col-17. However, double mutants between col-17 and other two components of this pathway col-15 and gna-1 were not obtained. -The epistasis analysis showed that frost is a very strong epistatic mutation; frost mutation was epistatic to 10 morphological mutants and 2 of them could be grouped into common developmental pathway (Fig. 38 pathway 2). It was interesting that all mutations hypostatic to the frost mutation were not correctable by Ca 2 + . Morever, some of them had slower growth on medium supplemented with exogenous Ca (i.e. gran, smco-8, spco-4 and mcb). 3.4.5. Interesting Interactions of cot-1 Mutant Some cot-1 double mutants had unexpected phenotypes. The cot-1 mutation is conditional and has wild type growth at 25 °C and restricted growth at the non-permissive (above 32 °C) temperature. According to this property, the phenotype of the 89 cot-1 double mutant grown at 25 °C should be the phenotype of the other mutant parent. However, double mutants cot-1 ;fr (Fig.60), smco-8 ;cot-\ (Fig.63), and cot-1; col-17 (Fig. 147) had very restricted growth at 25 °C, more restricted than that of both parents.. Shifted to non-permissive temperature these double mutants did not change growth rates and hyphal morphologies. This probably suggests that cot-1 lacks some functions of the wild type at permissive temperature. Probably wild type COT-1 protein needs functional FROST, SMCO-8, and COL-17 proteins to carry out its function. 90 Table 9. Conidiation of double mutants and their single mutant parents, (dm) double mutant, (+)conidial, (-) aconidial, (*) conidial, only in the presence of light cross double mutant dm parent 1 parent 2 # conidia conidia conidia 3 smco-6; col-1 + - -7 spco-11; col-1 - - -8 spco-11; col-8 - - -10 spco-4; col-1 - - -13 smco-6; col-8 - - -29 spco-11; col-15 + - + 31 smco-6; col-15 - - + 33 spco-4; col-15 + - + 38 smco-5; col-17 + + + 39 smco-6 col-17 - - + 41 col-15; spco-9 - + -46 spco-11; col-17 - - + 51 spco-4; pk - + * 53 smco-6; fr - - -56 spco-4; fr - - -63 col-15; smco-7 + + + 64 smco-8; col-15 - - + 72 smco-4; col-17 - + + 73 smco-7; col-17 - + + 81 smco-8; pk - - + * 86 col-4; smco-1 - + + 89 smco-7; col-4 - + + 90 spco-4; col-4 - - + 94 col-4; spco-9 + + -95 col-4; spco-10 + + + 96 col-4; spco-11 - + -97 col-4; spco-12 - + -98 col-16; spco-6 + + + 101 col-16; spco-10 + + + 104 col-16; smco-1 - + + 106 col-16; smco-5 - • + + 108 smco-7; col-16 - + + 109 col-16; spco-5 + + -110 col-8; col-17 - - + 111 smco-8; smco-6 - - -113 smco-8; spco-5 - - -114 spco-11; spco-10 + - + 120 col-4; fr - + -122 col-16; fr + + -127 col-16; pk - + + * 129 spco-6; smco-9 - + -91 141 smco-9; col-16 - - + 143 smco-8; fr - - -144 gran; fir - + * -147 spco-4; gran - - + * 149 spco-6; gran - + + * 159 col-4; gran - + + * 161 col-16; gran - + + * 163 smco-7; fr - + -164 gran; col-17 - + * + 166 gran; col-1 - + * -167 spco-11; gran - - + * 169 sn fr - + -180 spco-4 col-17 - - + 181 col-4; col-17 + + + 184 smco-7; cot-1 - + + 185 cot-1; gran + * + + * 186 spco-4; cot-1 - - + 187 mcb; col-17 - + + 189 col-4 cot-1 + + + 194 mcb; spco-4 - + -195 smco-7; cr-1 - + + 197 cot-1; fr - + -199 mcb; fr - + -208 spco-6; fr - + -210 smco-8 col-4 - - + 211 col-15; spco-7 - + + 216 smco-7; gna-1 - + + 218 col-15 gna-1 - + + 219 mcb; cr-1 + + + 220 smco-7; gran - + + * 224 spco-4; smco-7 - - + 227 spco-4; gna-1 - - + 228 col-4; gna-1 + + + 230 col-16; col-4 - + + 232 mcb; cot-1 + + + 238 col-16 gna-1 + + + 241 cot-1; col-16 - . + + 242 cot-1; col-17 - + + 244 col-16; mcb + + + 245 smco-8; smco-7 + - + 246 scr; fr - + -247 ipa fr - + -248 smco-8; spco-4 - - -249 smco-8 cot-1 + - + 250 smco-7 mcb - + + 253 spco-12 fr - - -254 cot-1; gna-1 + + + 92 255 coil; fr - + -256 smco-5 fr - + -257 spco-11 ;gna-l - - + 258 cr-l;cot-l + + + 261 col-15 col-16 - + + 262 gran;cy - + * + 273 col-4; spco-6 - + + 283 smco-6; col-4 - - + 289 smco-6 ; col-16 - - + 295 col-4; spco-5 + + -310 smco-4; col-16 -• + + 351 col-15; fr - + -359 gran; spco-12 - + * -375 sp ;fr - + * -93 Table 10. Growth rates of double mutants (dm) and their single mutants parents on Vogel's minimal medium. cross double mutants dm parent 1 parent 2 # parent 1, parent 2 mm/h. mm/h \ mm/h 3 smco-6; col-1 0.08 0.28 0.11 7 spco-11; col-1 0.09 0.51 0.11 8 spco-11; col-8 0.13 0.51 0.12 10 spco-4; col-1 0.11 0.58 0.11 13 smco-6; col-8 0.12 0.28 0.12 29 spco-11; col-15 0.11 0.51 0.09 31 smco-6; col-15 0.07 0.28 0.09 33 spco-4; col-15 0.12 0.58 0.09 38 smco-5; col-17 0.34 1.89 0.91 39 smco-6 col-17 0.10 0.28 0.91 41 col-15; spco-9 0.18 0.09 0.68 46 spco-11; col-17 0.18 0.51 0.91 51 spco-4 ; pk 0.21 0.58 0.08 53 smco-6; fr 0.18 0.28 0.82 56 spco-4 ;fr 0.49 0.58 0.82 63 col-15; smco-7 0.11 0.09 0.82 64 smco-8; col-15 0.41 0.88 0.09 72 smco-4; col-17 0.25 0.94 0.91 73 smco-7; col-17 0.35 0.82 0.91 81 smco-8; pk 0.09 0.88 0.08 86 col-4; smco-1 0.13 0.61 0.71 89 smco-7; col-4 0.39 0.82 0.61 90 spco-4; col-4 0.31 0.58 0.61 94 col-4; spco-9 0.43 0.61 0.68 95 col-4; spco-10 0.23 0.61 0.72 96 col-4; spco-11 0.02 0.61 0.51 97 col-4; spco-12 0.07 0.61 0.08 98 col-16; spco-6 0.21 0.55 0.23 101 col-16; spco-10 0.60 0.55 0.72 104 col-16; smco-1 0.22 0.55 0.71 106 col-16; smco-5 0.32 0.55 1.89 108 smco-7; col-16 0.31 0.82 0.55 109 col-16; spco-5 0.38 0.55 0.24 110 col-8; col-17 0.08 0.12 0.91 111 smco-8; smco-6 0.14 0.88 0.28 113 smco-8; spco-5 0.09 0.88 0.24 114 spco-11; spco-10 0.47 0.51 0.72 120 col-4; fr 0.32 0.61 0.82 122 col-16; fr 0.29 0.55 0.82 127 col-16; pk 0.06 0.55 0.08 129 spco-6; smco-9 0.10 0.23 0.12 141 smco-9; col-16 0.19 0.12 0.55 94 143 smco-8; fr 0.31 0.88 0.82 144 gran; fr 0.30 1.46 0.82 147 spco-4; gran 0.52 0.58 1.46 149 spco-6; gran 0.84 0.23 1.46 159 col-4; gran 0.39 0.61 1.46 161 col-16; gran 0.06 0.55 1.46 163 smco-7; fr 0.01 0.82 0.82 164 gran; col-17 0.05 1.46 0.91 166 gran; col-1 0.06 1.46 0.11 167 spco-11; gran 0.78 0.51 1.46 169 sn fr 0.12 0.44 0.82 180 spco-4 col-17 0.37 0.58 0.91 181 col-4; col-17 0.57 0.61 0.91 184 smco-7; cot-1 1.15 0.82 1.96 185 cot-1; gran 1.51 1.96 1.46 186 spco-4; cot-1 0.56 0.58 1.96 187 mcb; col-17 0.03 0.69 .0.9.1 189 col-4 cot-1 0.69 0.61 1.96 194 mcb; spco-4 0.54 0.69 0.58 195 smco-7; cr-1 0.28 0.82 0.33 197 cot-1; fr 0.01 1.96 0.82 199 mcb; fr 0.55 0.69 0.82 208 spco-6; fr 0.09 0.23 0.82 210 smco-8; col-4 0.13 0.88 0.61 211 col-15; spco-7 0.18 0.09 0.32 216 smco-7; gna-1 0.61 0.82 1.33 218 col-15 gna-1 0.08 0.09 1.33 219 mcb; cr-1 0.52 0.69 0.33 220 smco-7; gran 1.62 0.82 1.46 224 spco-4; smco-7 0.39 0.58 0.82 227 spco-4; gna-1 0.52 0.58 1.33 228 col-4; gna-1 0.58 0.61 1.33 230 col-16; col-4 0.51 0.55 0.61 232. mcb; cot-1 0.62 0.69 1.96 238 col-16 gna-1 0.52 0.55 1.33 241 cot-1; col-16 0.32 1.96 0.55 242 cot-1; col-17 0.06 1.96 0.91 244 col-16; mcb 0.57 0.55 0.69 245 smco-8; smco-7 0.16 0.88 0.82 246 scr; fr 0.45 0.97 0.82 247 ipa fr 0.87 1.33 0.82 248 smco-8; spco-4 0.22 0.88 0.58 249 smco-8 cot-1 0.21 0.88 1.96 250 smco-7 mcb 0.78 0.82 0.69 253 spco-12 fr 0.05 0.08 0.82 254 cot-1; gna-1 1.49 1.96 1.33 255 coil; fr 0.69 1.34 0.82 95 256 smco-5 fr 0.78 1.89 0.82 257 spco-ll;gna-l 0.66 0.51 1.33 258 cr-l;cot-l 0.31 0.33 1.96 261 col-15 col-16 0.08 0.09 0.55 262 gran;cy 0.98 1.46 1.03 273 col-4; spco-6 0.34 0.61 0.23 283 smco-6; col-4 0.62 0.28 0.61 289 smco-6 ; col-16 0.12 0.28 0.55 295 col-4; spco-5 0.14 0.61 0.24 310 smco-4; col-16 0.24 0.94 0.55 351 col-15; fr 0.06 0.09 0.82 359 gran; spco-12 0.23 1.46 0.08 375 sp ;fr 0.29 0.71 0.82 96 Figure 64. Hyphal morphology of wild type, A : vegetative hyphae, B: aerial hyphae 122 B Figure 66. Morphology of spco-4, A: vegetative hyphae, B: aerial hyphae 124 Figure 67. Hyphal morphology of col-17 A: vegetative hyphae, B : aerial hyphae 125 Figure 68. Hyphal morphology of cot-1 ;fr, A: vegetative hyphae, B: aerial hyphae 126 127 C H A P T E R I V 4. Response of Morphological Mutants to Exogenously Added Ca 2 + and M n 2 + 4.1. Introduction Several genes of the Neurospora crassa calcium signal transduction pathway have been cloned. These include genes coding for calmodulin (Capelli et al., 1993), the calcineurin catalytic subunit cna-1 (Higuchi et al., 1991), the calcineurin regulatory subunit cnb-1 (Kothe and Free, 1998), frost (Sone and Griffiths, 1999) and spray (Bok et al., 2001). Two of those mutants, frost and spray, were known to be correctable to wild type by exogenously added C a 2 + (Dicker and Turian, 1990; Sone and Griffiths, 1999). In addition the frost gene is involved in M n 2 + homeostatis and the frost mutant is sensitive to exogenous M n 2 + (Sone and Griffiths, 1999). The epistasis analysis ( Chapter HI) revealed several genes that interact with the frost gene; this raised the possibility that these other genes might also be involved in C a 2 + and M n 2 + homeostasis. 4.2. Material and Methods A l l mutant strains were grown in Vogel's medium supplemented with Ca and M n . Supplementary 100 m M C a 2 + and 5 m M M n 2 + as chlorides were added to the Vogel's minimal medium after autoclaving, as sterile aqueous solutions. According to individual response of each single mutant these concentrations were changed. For photographing hyphae, mutant strains were grown on cellophane. An autoclaved disk of cellophane was placed on the surface of the agar medium. An inoculum, either conidia or a mycelial block, was placed on the cellophane. The photographs were taken after 24 or 48 hours. Images of colonies were taken by Nikon COOLPIX500 digital camera. 128 4.3. Results A l l the morphological mutants used in this study have been screened for their response to exogenously added 100 m M C a 2 + and 5 mM M n 2 + . The results of this study are summarized in Table 12. Depending on the individual responses of each single mutant, other concentrations were also assayed. For all mutants except sn higher concentrations was also tested. Mutants sn (Fig.70), smco-1 (Fig.71) and smco-6 (Fig.72) were corrected to wild type either by exogenously added C a 2 + or M n 2 + . The mutant phenotype of sn can be corrected to wild type by 50-200 m M CaCl 2 or 8 mM M n C l 2 . Also, addition of 100-200 m M CaCh or 5 and 14 m M MnCb can correct mutant phenotypes of smco-1 and smco-6 respectively. 2+ Exogenously added Ca improved hyphal morphologies and growth rates of col-15 (Fig.73) and col-12 (Fig.74) but they were not corrected to wild type. A concentration of 5mM M n C l 2 increased the growth rates of smco-8 (Fig.75), and gran (Fig.76) mutants, although their hyphal morphologies were not significantly improved. It is interesting that the smco-7, smco-8 and gran mutants have slower growth on medium supplemented with Ca 2 + . Grown on medium supplemented with either C a 2 + or M n 2 + spco-11 did not form dichotomous branches (Fig.77). Mutants col-4, spco-4 and col-17 (Fig.7'8) showed slower growth either on medium supplemented with C a 2 + or M n 2 + . Several mutants did not show any response to either C a 2 + or M n 2 + ( Table 12). Wild type did not show any difference in size or appearance of hyphae or colony on medium supplemented with 1-5 mM M n C l 2 (Fig. 79 C). In fact, concentrations of 1-2 mM M n 2 + slightly increased the growth rate of the wild type. The wild type showed slower growth on medium supplemented with 7 mM M n 2 + . A concentration of 100 m M of C a 2 + did not change the growth rate of the wild type (Fig.79 B). However the wild type grown on medium supplemented with 100 mM C a 2 + formed more aerial hyphae and conidia. Also, conidia 129 appeared after 24 hours that was earlier than in control. A concentration of 200 mM Ca: lowered branch interval and decreased growth rate of the wild type. Table 11. Response of morphological mutants to 100 mM Ca 2 + and 5 mM Mn (*) corrected to wild type, (•) greatly improved hyphal morphology, ( A ) decreased growth rate, (oo) slightly increased or decreased growth rate better growt l slower growth both Ca 2 + Mn 2 + both Ca 2 + Mn 2 + sn * fr * smco-8 • spco-4 A smco-8 A cr-100 smco-1 * sp * smco- 7 • col-4 A smco-7 A smco-6 * col-15 • gra n • col-17 A mcb A spcolloo . col-12 • mcb oo SpCo9 oo gran A gna-1 oo spco-6°° smco-5 00 spco-5°° smco-4 00 col-16oo spco-9 00 COt-loo spco-10 00 gna-1 00 cy 00 pk 00 Table 12. Morphological mutants that did not change morphology either on 100 m M C a 2 + or 5 m M M n 2 + or both. both Ca 2 + M n 2 + wild type cr-1 spco-5 smco-9 spco-10 smco-4 col-15 spco-7 col-12 spco-12 smco-4 col-1 smco-5 col-8 sp del pk coil fr ipa 130 Also some of double mutants obtained in this study that had one or both parents correctable with either C a 2 + or M n 2 + have been screened for their responses to these ions. The double mutants spco-4;fr, col-16;fr, smco-8;smco-6, smco-6;col-16 and col-4;smco-1 showed slower growth on medium supplemented with exogenous C a 2 + although each of these double mutants involving a C a 2 + correctable single mutation. Also, these double mutants did not show any response to exogenously added M n 2 + . It is interesting that the double mutant smco-8;smco-6 did not response to M n 2 + although both mutations are fully or partially correctable by M n 2 + . Neither exogenous C a 2 + nor M n 2 + changed branching patterns and growth rates of the double mutants smco-7;fr and col-4 ;gran. However, addition of C a 2 + or M n 2 + greatly improved either hyphal morphology or growth rate of double mutants gran;fr (Fig.80), smco-8;f (Fig.81), and cot-1 ;fr (Fig.82). 4.4. Discussion 4.4.1. Response of Single Mutants to Exogenous Ca 2 + and M n 2 + 2+ 2+ This analysis showed clear groups of mutants with different responses to Ca and Mn . It is interesting that the mutant phenotypes of sn, smco-1 and smco-6 have been corrected either by exogenous C a 2 + or M n 2 + . In all three cases, much smaller concentrations of M n 2 + were as effective as larger concentrations of Ca 2 + . It is known that calcineurin is regulated by the C a 2 + / calmodulin complex, and also that calcineurin is involved in M n 2 + uptake in yeast and requires M n 2 + for its functions (Rusnak and Mertz, 2000). Therefore, these three genes might be involved in the synthesis and activity of calcineurin. The mutants frost, spray and smco-6 exhibited a very similar growth pattern. In all three mutants the leading hyphae extended past the colony front with reduced frequency of branch formation and after a short period of extension, they subsequently resumed their 131 increased frequency of branching. It is interesting that most of the C a 2 + correctable mutants frost, smco-1, sn and col-12 belong to linkage group I. Several double mutants col-15;smco-6, smco-6;fr, sn;fr, col-15;fr, and sp;fr have been obtained from Ca2+correctable mutants; no one of them showed epistasis. It seems that all of them affect different pathways involved in C a 2 + signaling in Neurospora. This is not a surprise i f we take into account that many cellular processes are regulated by calcium. Crosses between smco-6 and sp, smco-6 and smco-1, smco-1 and fr did not yield perithecia. In crosses between smco-1 and sn, smco-1 and sp, smco-1 and col-15 and smco-6 and sn the double mutants did not survive probably due to synthetic lethality. These results may suggest that pathways where these genes act are essential for cell viability. 4.4.2. Response of Double Mutants to Exogenous Ca 2 + and M n 2 + The double mutant gran.fr responded to exogenous C a 2 + as was expected. The phenotype was that of gran grown on the same medium (Fig.80). The frost hyphal morphology of the double mutant gran;fr disappeared, obviously the frost mutation was overcome by the excess C a 2 + and the double mutant responded like the single mutant gran. Although, gran had better growth on medium supplemented with exogenous M n 2 + , the double mutant gran;fr did not show any improvement in hyphal morphology (Fig.80). The double mutant smco-8;fr was also interesting because the parent smco-8 responded to M n 2 + only and frost responded to C a 2 + only. It was expected that this double mutant grown on medium supplemented with C a 2 + would have the phenotype of smco-8 grown on the same medium. However, the hyphal morphology of the double mutant smco-8;fr (Fig.81) was corrected to wild type by addition of 400 mM Ca 2 + . This result probably suggests some suppression of smco-8 mutation. Grown on medium supplemented with 5 132 mM Mn the phenotype of the double mutant smco'-8;fr did not change significantly, hence although M n 2 + can correct smco-8 this does not occur in the presence of frost. There was an interesting response of the double mutant cot-1; fr to the elevated concentrations of exogenous Ca 2 + . The double mutant cot-1 ;fr had one of the most aberrant phenotypes obtained in this study; the hyphae lost their polar growth and the overall morphology was seriously altered. Polar hyphal growth was restored on medium supplemented with 400 mM C a 2 + (Fig. 82) and even a smaller concentration of Ca 2 + , 100 m M also greatly improved the hyphal morphology. Hyphae lost their bead-like appearance and many hyphal tips had essentially the wild type phenotype. This was unexpected as C a 2 + inhibited cot-1. Also, 7 mM M n 2 + greatly improved hyphal morphology and the growth rate of the double mutant cot-1 ;fr; bulbous cells disappeared and many hyphae had wild type appearance although M n 2 + did not affect either mutant individually. The distances between septa were long and in some hyphae they were longer than in the wild type. This observation may suggest a role of M n 2 + in polarized hyphal growth and inhibition of branching. The mutant phenotype of cot-1 ;fr was corrected by exogenous C a 2 + or M n 2 + , however neither of these ions corrected the mutant phenotype at a non-permissive temperature. Although hyphal morphologies and growth rates of the double mutants smco-8;fr (Fig.81), and cot-1 ;fr (Fig.82) were greatly improved, their colonies retained the frost appearance. 133 138 Figure 75. Hyphae of smco-8, A Vogel's medium, B Vogel's medium supplemented with 100 mM Ca2+, C Vogel's medium supplemented with 5 mM Mn2+ 139 Figure 76. Response of gran to exogenous Ca2+ and Mn2+ A: Vogel's medium, B: Vogel's medium supplemented with 5 mM Mn2 C: Vogel's medium supplmented with 100mMCa2+ 140 Figure 77. Hyphae of spco-11 A . Vogel's medium, B . Vogel's medium supplemented with 100 m M C a 2 + , C. Vogel's medium supplemented with 5 m M M n 2 + 141 Figure 78. Colonies of col-17 A: Vogel's medium, B: Vogel's mdium supplemented with 5 mMMn2+, C: Vogel's medium supplemented with 100 mM Ca2+ 142 Figure 82. Response of the double mutant cot-l;fr to exogenous Ca2+ and Mn2 A: Vogel's medium, B: Vogel's medium supplemented with 400 mM Ca^+, C: Vogel's medium supplemented with 7 mM Mn^+ 146 CHAPTER V 5. Integrated Discussion A n analysis of epistasis revealed interesting interactions between certain N. crassa morphological mutants and gave a provisional characterization of four pathways involved in hyphal tip growth and branching. The analysis showed five distinct groups of double mutants showing different types of interactions. The revealed interactions as well as a phenotypic characterization of those mutant loci will be useful in facilitating further studies in morphogenesis. This study pointed out several mutants, sn, smco-1, smco-6, col-12 and col-15, whose phenotypes were completely or partially corrected by either exogenous C a 2 + or M n 2 + . The dramatic morphological effects on the phenotypes of mutants smco-1, smco-6 and sn, brought about by the addition of exogenous C a 2 + and M n 2 + , may suggests that these ions are largely responsible for the slow extension rates and the abnormal morphologies seen in these three mutants. But, how exactly the observed effects caused by extremely high concentrations of C a 2 + and M n 2 + ions corrected the mutant phenotypes remains to be determined. The results reported in this study add support to models favoring an important role for C a 2 + in regulation of hyphal tip growth and branching. Furthermore, more surprisingly this report implicates M n 2 + in the physiology of tip extension in Neurospora and suggests that some of the morphological mutants of Neurospora crassa may be deficient in their processing of M n 2 + . The dramatic improvement of grossly altered hyphal morphology of the double mutant cot-l;fr by exogenous C a 2 + and M n 2 + may suggests involvement of these ions in a polarized hyphal growth. 5.1. Future Studies The data presented in this study have pointed to specific future experiments: 147 1. ) Crosses involving components of the provisional pathways characterized in this study, and some other morphological mutants may result in double mutants showing epistasis. This may allow addition of new components to those pathways. 2. ) This study showed that several double mutants from the group expressing a novel phenotype resembled some other morphological mutants that were not parental strains. This probably suggests interconnection of pathways where those genes act. It is also possible that other double mutants from this group resemble some other morphological mutants that were not used in this study. Characterization of existing morphological mutants may reveal such phenotypes. 3. ) Approximately one third of existing Neurospora morphological mutants were used in this study and several of them responded either to C a 2 + or M n 2 + or both; the screen showed several clear groups of mutants according to their response to these ions. A screen including the rest of existing morphological mutants should result in a similar grouping as presented in this thesis. 4. ) Results from my study showed the role of M n 2 + in hyphal tip growth and branching. Future experiments may clarify a specific role of M n 2 + in hyphal polarized growth and branching inhibition. 5. ) In N. crassa calcineurin A has been reported to be essential for hyphal growth and branching (Prokisch et al., 1997). 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Attempted crosses between morphological mutants, (dm) mutant, (+) double mutant was obtained; (-) double mutant was not obtained cross # cross d m cross # cross d m 1 smco-5 X col-1 - 186 spco-4 X cot-1 + 2 smco-1 X col-1 - 187 mcb X col-17 + 3 col-1 X smco-6 - 188 cr-1 X col-17 -4 col-1 X spco-7 - 189 col-4 X cot-1 + 5 spco-12 X col-1 - 190 gran X scr -6 spco-9 X col-1 - 191 cr-1 Xgran -7 spco-11 X col-1 + 192 mcb X gran -8 spco-i/ X col-8 + 193 col-15 X cr-1 -9 col-8 X spco-9 - 194 mcb X spco-4 + 10 spco-4 X col-1 + 195 smco-7 X cr-1 + 11 col-8 X fr - 196 spco-10 X fr -12 smco-9 X col-8 - 197 COt-1 X fir + 13 smco-6 X col-8 + 198 col-1 X cot-1 -14 smco-5 X col-8 - 199 mcb X fr + 15 spco-12 X col-8 - 200 spco-12 X gna-1 -16 col-12 X spco-6 - 201 col-4 X cy -17 sn X pk - 202 gran X smco-4 -18 smco-9 X smco5 - 203 mcb X col-4 -19 col-8 X spco-7 - 204 cr-1 Xfr -20 spco-4 X col-8 - 205 sn X gna-1 21 col-12 X spco-7 - 206 sn X coil -22 col-12 X smco-6 - 207 sn X sp -23 spco-4 X col-12 - 208 spco-6 X fir + 24 spco-6 X col-12 - 209 smco-5 X smco-8 -25 spco-9 X col-12 - 210 smco-8 X col-4 + 26 col-12 X spc-10 - 211 col-15 X spco-7 + 27 sn X spco-12 - 212 spco-9 X gna-1 -28 col-15 X smco-5 - 213 gna-1 X cr-1 -29 spco-11 X col-15 + 214 gna-1 X mcb -30 gran X sn - 215 gna-1 X smco-8 -31 col-15 X smco-6 + 216 gna-1 X smco-7 + 32 smco-9 X col-15 - 217 gna-1 X col-17 -33 col-15 X spco-4 + 218 gna-1 X col-15 + 34 spco-6 X col-15 - 219 mcb X cr-1 + 35 smco-1 X col-8 - 220 gran X smco-7 + 36 col-16 X spco-4 - 221 del X gran -37 spco-7 X col-17 - 222 ipa X smco-1 -38 smco-5 X col-17 + 223 smco-1 X scr -39 smco-6 X col-17 + 224 smco-7 X spco-4 + 40 col-15 X spco-10 - 225 gna-1 X fr -41 col-15 X spco-9 + 226 gna-1 X gran -42 col-15 X spco-12 - 227 gna-1 X spco-4 + 159 43 sn X spco-7 - 228 col-4 X gna-1 + 44 sn X spco9 - 229 ipa X smco-7 -45 smco-1 X pk - 230 col-16 X col-4 + 46 spco-11 X col-17 + 231 smco-7 X del -47 spco-10 X col-17 - 232 smco-7'X scr -48 sn X spco-10 - 233 col-1 X cot-1 -49 pk X spco-12 - 234 col-8 X cot-1 -50 cr-1 X smco-8 - 235 smco-8 X mcb -51 spco-4 X pk + 236 sn X spco-11 -52 pk X spco-7 - 237 cy X fr -53 smco-6 X /r 238 . col-16 X gna-1 + 54 spco-7 X /r - 239 smco-8 X cy -55 smco-9 X /r - 240 spco-7 X gna-1 -56 spco-4 X /r + 241 cot-1 X col-16 + 57 smco-4 X col-15 - 242 cot-1 X col-17 + 58 spco-7 X smco-5 - 243 mcb X cot-1 + 59 X smco-4 - 244 col-16 X mcb + 60 sn X co/-4 - 245 smco-7 X smco-8 + 61 M X / r - 246 scr X fr + 62 spco-5 X / r - 247 ipa X fr + 63 smco-7 X col-15 + 248 smco-8 X spco-4 + 64 smco-8 X col-15 + 249 smco-8 X cot-1 + 65 spco-5 X col-15 - 250 smco-7 X mcb + 66 smco-4 X co/-# - 251 smco-4 X gna-1 67 smco-7 X co/-# - 252 smco-1 X gna-1 -68 smco-4 X co/-/ - 253 spco-12 X fr + 69 smco-1 X col-12 - 254 gna-1 X cot-1 + 70 smco-6 X smco-5 - 255 coil X fr + 71 smco-4 X co/-4 - 256 smco-5 X fr + 72 col-17 X smco-4 + 257 spco-11 X gna-1 + 73 smco-7 X col-17 + 258 cot-1 X cr-1 + 74 smco-5 X spco-4 - 259 del X smco-1 -75 col-17 X smco-8 - 260 ipa X gran -76 smco-4 X col-12 - 261 col-15 X col-16 + 77 smco-7 X col-12 - 262 gran X cy + 78 smco-8 X col-12 - 263 cr-1 X spco-12 -79 spco-5 X col-12 - 264 cr-1 X spco-11 -80 smco-1 X /r - 265 del X smco-8 -81 p& X smco-8 + 266 cr-1 X smco-1 -82 spco-9 X /r - 267 gna-1 X smco-6 -83 smco-4 X /r - 268 mcb X ipa -84 spco-11 X pk - 269 mcb X coil -85 X smco-7 - 270 cr-1 X spco-10 -86 col-4 X smco-1 + 271 cr-1 X spco-9 -87 col-4 X smco-5 - 272 cr-1 X spco-7 -88 smco-6 X /?& - 273 col-4 X spco-6 + 89 smco-7 X co/-4 + 274 cr-1 X col-4 -160 90 col-4 X spco-4 + 275 cr-1 X spco-6 -91 col-17'X col-16 - 276 cr-1 X spco-5 -92 smco-9 X pk - 277 scr X col-16 -93 col-4 X spco-7 - 278 col-1 X fr -94 col-4 X spco-9 + 279 cot-1 X col-12 -95 col-4 X spco-10 + 280 ipaX col-15 -96 col-4 X spco-11 + 281 cy X col-15 -97 col-4 X spco-12 + 282 scr X col-15 -98 col-16 X spco-6 + 283 col-4 X smco-6 + 99 col-16 X spco-7 - 284 cr-1 X spco-4 -100 col-16 X spco-9 - 285 col-15 X del -101 col-16 x spco-10 + 286 spco-11 X gna-1 -102 col-16 X spco-11 - 287 smco-1 X cot-1 -103 col-16 X spco-12 - 288 gna-1 X spco-10 -104 col-16 X smco-1 + 289 smco-6 X coir 16 + 105 smco-5 X col-12 - 290 .: smco-6 X sp -106 col-16 x smco-5 + 291 smco-6 X gran -107 smco-5 X pk - 292 smco-4 X cr-1 -108 smco-7' x col-16 + 293 smco-7 X sp -109 col-16 X spco-5 + 294 smco-5 X cr-1 -110 col-8 X co/-/7 + 295 col-4 X spco-5 + 111 smco-8 X smco-6 + 296 smco-8 X col-8 -112 smco-1 X spco-4 - 297 smco-9 X col-1 -113 smco-8 X spco-5 + 298 smco-9 X col-4 -114 spco-11 X spco-10 + 299 spco-5 X col-17 -115 col-12 X - 300 spco-6 X col-17 -116 col-15 X - 301 spco-9 X pk -117 col-16 X s/? - 302 spco-10 X col-8 -118 col-17 x s/? - 303 del X col-17 -119 X spco-5 - 304 spco-10 X pk -120 col-4 X / r + 305 spco-10X sp -121 cr-/ X col-16 - 306 spco-10 X gran -122 col-16 X /r + 307 spco-11 X col-12 -123 co/-/7 X /r - 308 cot-1 X smco-1 -124 X - 309 spco-12 X col-12 -125 p& X col-12 - 310 smco-4 X col-16 + 126 pk X col-15 - 311 spco-11 X mcb -127 pk X col-16 + 312 smco-8 X col-1 -128 gran X col-15 - 313 sp X scr -129 spco-6 X smco-9 + 314 gna-1 X col-8 -130 sp X spco-4 - 315 scr X spco-4 -131 sp X spco-7 - 316 cr-1 X col-8 -132 sp X spco-11 - 317 coil X ipa -133 sp X spco-12 - 318 gna-1 X smco-5 -134 sp X co/-4 - 319 coil X scr -135 5/7 X smco-1 - 320 mcb X smco-1 -136 sp X smco-4 - 321 mcb X smco-6 -161 137 col-1 X sn - 322 spco-4 X ipa -138 sp X smco-8 - 323 scr X ipa -139 sp X smco-9 - 324 mcb X smco-5 -140 col-16 X smco-8 - 325 scr X mcb -141 smco-9 X col-16 + 326 coil X smco-8 -142 pk X col-17 - 327 smco-5 X coil -143 smco-8 x fr + 328 cr-1 X smco-9 -144 gran X fr + 329 col-4 X del -145 spco-5 X sn - 340 sp X mcb -146 smco-9 X gran - 341 col-15 X col-12 -147 spco-4 X gran + 342 ipa X smco-8 -148 spco-7 X gran - 343 scr X smco-8 -149 spco-6 X gran + 344 coil X spco-4 -150 spco-9 X gran - 345 col-8 X cr-1 -151 pk X gran - 346 cot-1 X spco-11 -152 sp X gran - 347 cot-1 X spco-7 -153 sn X col-8 - 348 col-4 X coil -154 sn X col-12 - 349 ipa X smco-5 -155 sn X col-15 - 350 spco-11 X ipa -156 smco-1 X gran - 351 col-15 X fr + 157 smco-5 X gran - 352 del X fr -158 smco-8 X gran - 353 col-8 X mcb -159 col-4 X gran + 354 cr-1 X scr -160 sn X smco-1 - 355 mcb X spco-5 -161 col-16 X gran + 356 smco-1 X col-15 -162 col-8 X gran - 357 sp X spco-9 -163 smco-7 X fr + 358 sn X spco-6 -164 gran X col-17 + 359 gran X spco-12 + 165 sn X col-16 - 360 cr-1 X ipa -166 gran X co/-/ + 361 gna-1 X coil -167 spco-11 X gra« + 362 gna-1 X cy -168 sn X col-17 - 363 col-4 X scr -169 X fr + 364 mcb X col-1 170 sn X smco-1 - 365 mcb X sn -171 spco-11 X /r - 366 sn X scr -172 col-12 X - 367 sn X spco-4 -173 5 « X smco-6 - 368 scr X smco-5 -174 smco-8 X - 369 coil X gran -175 s« X smco-4 - 370 cot-1 X col-15 -176 col-12 X /r - 371 pk X mcb -177 X smco-5 - 372 pk X cr-1 -178 sn X smco-7 - 373 ipaX col-17 -179 col-17 X spco-9 - 374 col-17 X col-15 -180 spco-4 X col-17 + 375 sp X fr + 181 col-4 X co/-77 + 376 smco-8 X col-8 -182 co/-/ X co/-/7 - 377 coil X col-17 -183 X smco-9 - 378 col-17 X scr -162 184 smco-7 X cot-1 + 379 c y X col-17 -185 cot-1 X gran + 380 smco-1 X smco-6 -381 . col-4 X ipa -163 186 189 200 204 222 

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