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Characterization of scytalone dehydratase and reductase genes and expression of melanin biosynthesis… Wang, Honglong 2002

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CHARACTERIZATION OF SCYTALONE DEHYDRATASE AND REDUCTASE GENES AND EXPRESSION OF MELANIN BIOSYNTHESIS GENES IN OPHIOSTOMA FLOCCOSUM by  HONGLONG WANG  B.Sc, Tianjin Normal University, 1986 M.Sc., Tianjin Normal University, 1989  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENT FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY  in  THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Wood Science, Faculty of Forestry)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A July, 2002 © Honglong Wang, 2002  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  for  his  this  and  scholarly  or  thesis  study.  her  for  of  Uioficl  T h e U n i v e r s i t y o f British Vancouver, Canada  Date  DE-6  (2/88)  s</  7  S*>'&>«JL  Columbia  ' 2 .  I further  gain  requirements that  agree  may  be  It  is  representatives.  financial  the  1 agree  purposes  permission.  Department  of  shall  not  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  head  make  it  extensive of  my  copying  or  my  written  Abstract Wood sapstain is a significant economic problem for the lumber industry. The discoloration of sapwood is mainly caused by sapstain fungi, which grow on wood and produce dark or brown pigment. The aim of the thesis was to obtain some molecular information about the pigmentation of sapstain fungi by cloning and characterizing the major melanin genes. A transformation system is the prerequisite for conducting both gene disruption and genetic complementation of an organism. Transformation systems were set up for Ophiostoma floccosum 387N and other major sapstain fungal species such as Ophiostoma piceae using the transformation vectors p A N 7 - l and pCJ31004. This transformation system was applied to attempt to disrupt the cloned genes, THN1 encoding a melanin pathway reductase gene and OSD1 encoding a scytalone dehydrates gene in 387N. Unfortunately, no disruptant was identified by screening more than 2,000 transformants. We concluded that homologous D N A integration in O. floccosum 378 would be a rare event. We isolated and characterized a putative scytalone dehydratase gene (OSD1) from O. floccosum 387N encoding a predicted polypeptide sequence of 216 amino acids that shared high homology to other fungal melanin scytalone dehydratases. The function of OSD1 was determined by complementing a Colletotrichum lagenarium scytalone dehydratase deficient mutant. OSD1 was able to restore the melanization and pathogenicity of the mutants. A reductase gene (THN2) encoding a protein of 284 amino acids was isolated, and it shared a 44% amino acid identity to the O. floccosum THN1 genes' deduced protein sequence. We confirmed the function of the THN2 gene by complementing the D H N melanin deficient, non-pathogenic mutants of C. lagenarium and Magnaporthe grisea that lack the 1,3,8-trihydroxynaphthalene reductase gene. Sequence analysis of all available fungal melanin reductases showed that two groups of the reductases are present in fungal D H N melanin biosynthetic pathway. THN1 and THN2 belonged to different groups. We tried to complement a double mutant of M. grisea, where the 1,3,6,8-tetrahydroxynaphthalene reductase gene and the 1,3,8trihydroxynaphthalene reductase gene have been knocked out, using THN1, THN2 and the combination of THN1 and THN2, respectively. The results indicated that both reductases can not function as the 1,3,6,8-tetrahydroxynaphthalene reductase. However, whether they function in a similar way in O. floccosum remains unknown. A partial melanin PKS gene (OPKS1) was cloned in O. floccosum. The expression of the melanin genes, OPKS1, THN1, OSD1 and THN2 was associated with the mycelial differentiation and affected by nutrients.  ii  Table of Contents Abstract  ii  Table of Contents  iii  List of Figures  v  List of Tables  viii  List of Abbreviations  ix  Acknowledgements  xiv  Chapter 1 Introduction  1  1.1  Fungal melanin types  1  1.2  Molecular genetics of D H N melanin biosynthesis  7  1.3  Inhibitors of D H N melanin biosynthetic pathway  18  1.4  Fungal melanin functions  20  1.5  Sapstaining fungi and melanin  23  1.6  Research objectives  26  Chapter 2  Transformation of three Ophiostoma species and tentative genetic disruption of O. floccosum melanin genes  29  2.1  Introduction  29  2.2  Material and methods  32  2.3  Results  40  2.4  Discussion  56  Chapter 3  3.1  Isolation and characterization of an O. floccosum OSD1 gene that complements a Colletotrichum lagenarium melanin-deficient mutant  Introduction.  61 61  iii  3.2  Material and methods  63  3.3  Results  71  3.4  Discussion  86  Chapter 4 Function analysis of the second melanin reductase gene in O. floccosum 387N  91  4.1  Introduction  91  4.2  Material and methods  93  4.3  Results  97  4.4  Discussion  Chapter 5  112  Cloning a partial PKS gene and analyzing transcriptional patterns of melanin genes in O. floccosum 387N  122  5.1  Introduction  122  5.2  Material and methods  124  5.3  Results  128  5.4  Discussion  135  Chapter 6  Conclusions and future work  142  6.1  Conclusions  142  6.2  Future directions  144  References  Appendix  147  Fungal DHN melanin gene system and modeling  iv  163  List of Figures  Figure 1.1.  D O P A melanin biosynthesis  3  Figure 1.2.  G D B H melanin biosynthesis  4  Figure 1.3.  Catechol melanin biosynthesis  6  Figure 1.4.  D H N melanin biosynthesis  6  Figure 2.1.  The construction of the THN1 disruption vector pANA476  36  Figure 2.2.  The construction of the OSD1 disruption vector pBSD  37  Figure 2.3.  Yeast cells and protoplasts of O. floccosum  Figure 2.4.  P C R analysis of Ophiostoma transformants  Figure 2.5.  Dot blot analysis of Ophiostoma transformants  Figure 2.6.  The assumed integration event by homologous recombination of the  41 :  47 48  pANA476 into genomic 387N D N A  50  Figure 2.7.  P C R screening pANA476 transformed O. floccosum transformants  50  Figure 2.8.  The assumed integration event by homologous recombination of the pBSD into genomic 387N D N A  53  Figure 2.9.  P C R screening pBSD transformed O. floccosum transformants  53  Figure 2.10.  Novo-PCR with Triton-100  54  Figure 2.11.  Novo-PCR with D M S O  55  Figure 2.12.  Southern hybridization of the O. floccosum transformants with pBSD.. ..55  Figure 3.1.  A n amino acid sequence alignment of the scytalone dehydratases of C. lagenarium a n d M grisea PCR products of O. floccosum genomic D N A using SD 1/SD2 as primers PCR products amplified with plasmid D N A from bacterial Transformants with the primer combinations of SD1/SD2  Figure 3.2. Figure 3.3.  67 72 72  Figure 3.4.  Figure 3.5.  D N A sequences and deducted amino acid sequence of the 420-bp P C R Product  74  P C R products amplified with 387N genomic library aliquots with the nested primer combinations SD5/SD6  75  Figure 3.6.  Identification of the D N A fragment containing OSD1  75  Figure 3.7.  The construction of the vector pBSU2  76  Figure 3.8. Figure 3.9.  Nucleotide and deduced amino acid sequences of the scytalone dehydratase from O. floccosum Comparison of the deduced amino acid sequence of O. floccosum  78  OSD1 with those of C. lagenarium, M. grisea, and A. fumigatus  81  Figure 3.10.  The construction of the transformation vector, pESD  83  Figure 3.11.  The colour of C. lagenarium cultures  Genomic Southern blot analysis of the C. lagenarium Scd" mutant transformed with O. floccosum OSDlgene carrying plasmid pESD Figure 3.13. RT-PCR detection of the O. floccosum OSD1 gene transcript from the C. lagenarium Scd" mutant transformed with the O. floccosum OSD1 gene  83  Figure 3.12.  Figure 3.14.  Figure 4.1.  Pathogenicity test of the melanin-restored transformant Complemented with O. floccosum OSD1 gene  84  85  85  A nucleotide sequence comparison of the two insert sequences of the plasmids, pGT2 and pGT5 using the BLAST2 program  98  Figure 4.2.  The P C R product of genomic 387 using THN3 and THN4 as primers  98  Figure 4.3.  Identification of the D N A fragments containing THN2  99  Figure 4.4.  Nucleotide and deduced amino acid sequences of the 101  O. floccosum THN2 Figure 4.5.  The relationships of fungal melanin reductases  105  Figure 4.6.  The colour of the C. lagenarium and M. grisea cultures  106  Figure 4.7.  Appressorium pigmentation of the melanin-restored vi  Figure 4.8.  Figure 4.9.  Figure 4.10.  Figure 4.11.  transformants o f C . lagenarium  107  Appressorium pigmentation of the melanin-restored transformants of M. grisea  107  Pathogenicity test of melanin restored transformants of C. lagenarium, complemented with O. floccosum THN2  108  Genomic Southern blot analysis of the C. lagenarium and M. grisea mutants transformed with O. floccosum THN2 gene  108  RT-PCR detection of the O. floccosum THN2 gene transcript from the C. lagenarium and M. grisea mutants transformed with the O. floccosum THN2 gene  110  Figure 4.12.  The colour of the M. grisea double mutant cultures  Figure 4.13.  Genomic Southern blot analysis of the M. grisea double mutant transformed with O. floccosum THN1, THN2 and both  Figure 4.14.  Figure 4.15. Figure 5.1.  Figure 5.2.  ,  RT-PCR detection of the O. floccosum reductase gene transcripts from the M. grisea double mutants transformed with the O. floccosum THN1, THN2 and both  110  Ill  112  Comparison of the deduced amino acid sequence of fungal reductases... 115 Nucleotide and deduced amino acid sequences of O. floccosum OPKS1  129  Alignment of the p-ketoacyl synthase motif of the cloned OPKS1 PCR product with other PKS ketosynthase motifs  130  Figure 5.3.  The relationships of the P-ketoacyl synthase motif of fungal PKSs  131  Figure 5.4.  RT-PCR detection of the transcripts of the four melanin genes in conidia, yeast cells and mycelium of O. floccosum Time course of expression of the OPKS1, THN1, OSD1 and THN2 genes during the growth of O. floccosum on media 2  Figure 5.5. Figure 5.6.  The expression of the OPKS1, THN1, OSD1 and THN2 genes during the growth of O. floccosum on B-media supplemented with different combinations of carbon and nitrogen sources  vii  133 134  135  List of Tables Table 2.1.  The relation among OD oo values, yeast-like cell budding rates, and protoplasting rates of O. floccosum 387N cultured in proline media  Table 2.2.  Production of protoplasts of O. floccosum 387N using different enzyme concentrations and digestion time  42  The effects of P E G concentrations on transformation rates of O. floccosum 387N transformed with different amounts of p A N 7 - l DNA  45  The effects of D N A amount on the transformation rates of O. floccosum, O. piceae and O. quercus isolates transformed withpAN7-l orpCB1004  46  Different phenotypes of the transformants transformed using the OSD1 disruption vector pBSD  52  List of the oligonucleotides synthesized for use in sequencing and as PCR primers  65  Inferred amino acid composition of O. floccosum OSD1 deduced protein  80  Inferred amino acid composition of O. floccosum THN2 deduced protein  103  Table 2.3.  Table 2.4.  Table 2.5. Table 3.1. Table 3.2. Table 4.1. Table 4.2.  6  Amino acid sequence identity matrix (%) of fungal reductases of O. floccosum THN1 and THN2 with C. lagenarium THR, M. grisea 3HNR and 4HNR, A. alternata BRM2, C. heterostrophus Brnl and A. fumigatus arp2  Table 5.1.  List of the oligonucleotides synthesized for PCR reactions  Table 5.2.  The color of the O. floccosum cultures on different nutrient media  viii  42  104 125 132  List of Abbreviations  5', 3'  denotes 5' -hydroxy or 3' -phosphate end of sequence  AaTHN  A. alternata 1,3,8-THN reductase  A , C. G, T  nucleotides adenosine, cytosine, guanosine, thymidine  ACP  acyl carrier protein  AfTHN  A. fumigatus 1,3,6,8-THN reductase  amp  ampicillin  AT  acetyl/malonyl transferase domain  B  brown  BC  British Columbia  bp  base pair  BSA  bovine serum albumin  cDNA  complementary deoxyribonucleic acid  ChTHN  C. heterostrophus 1,3,8-THN reductase  Ci  Curie  C1THN  C. lagenarium 1,3,8-THN reductase  CM  complete medium  DB  dark brown  DDAC  didecyl dimethyl ammonium chloride  DDN  3,4-dihydro-4,8-dihydroxy-l naphthalenone  DHN/1,8-DHN  dihydroxynaphthalene  DMSO  dimethyl sulfoxide  DNA  deoxyribonucleic acid  ix  DNase  deoxyribonuclease  dNTP  deoxyribonucleoside triphosphate  DOPA  dihydroxyphenylalanine  EDTA  ethylene diamine tetra acetic acid  est  Expression sequence tag  EtBr  ethidium bromide  EtOH  ethanol  E-Value  Expected value  GDHB  glutaminyl-3,4-dihydroxybenzene  GHB  y-glutammyl-4-hydroxybenzene  h  hour  4HNR  M. grisea 1,3,6,8-THN reductase  hph  hygromycin phosphotransferase  HR  ratio of hydrophilicity to hydrophobicity of a protein  IR  infrared  kb  kilobase  kDa  kilodalton  KS  (3-ketoacyl synthase domain  LB  Light Brown  u.  micro  m  milli  MEA  malt extract agar  MgTHNA/T4HN  M. grisea 1,3,6,8-THN reductase  x  MgTHNB/T3HN  M. grisea 1,3,8-THN reductase  min  minute(s)  minus-RT  R N A samples without reverse transcriptase treatment  mm  millimeters  mRNA  messenger ribonucleic acid  MSAS  6-methylsalicylic acid synthase  NMR  nuclear magnetic resonance  NCBI  National Center of Biotechnology Information  No.  number  Novo-PCR  Novozym-aid PCR  nr  non-redundant  OD600  optical density at 600 nm  OfTHNA  O. floccosum 1,3,6,8-THN reductase  OfTFfNB  O. floccosum 1,3,8-THN reductase  ORF  open reading frame  PCR  polymerase chain reaction  PCP  polychlorophenates  PEG  polyethylene glycol  pfu  plaque-forming unit(s)  PM  proline medium  PKS  polyketide synthase  PDA  potato dextrose agar  PS  potato dextrose broth  xi  PSA  potato sucrose agar  RM  regeneration medium  rDNA  D N A coding for rRNA  RNase  ribonuclease  rRNA  ribosomal  RNA  rpm  round per minute  S  protein solubility  SADH  short chain alcohol dehydrogenase  SD  scytalone dehydratase  SDS  sodium dodecyl sulfate  sp  species  SSC  0.15 M NaCl/0.015 M Na3_citrate, pH 7.6  STC buffer  1 M sorbitol, 50 m M CaCl , 25 m M Tris-HCl, pH 7.5  TAE  Tris-acetate E D T A  TC buffer  50 m M CaCl , 2.5 m M Tris-Cl, pH 7.5  TCMM  2-(2-thiocyanomethylthio) benzothiozole  TE buffer  10 m M Tris-HCl pH 7.5; 1 m M E D T A  2  2  1,3,6,8-THN  1,3,6,8-tetrahydroxynaphthalene  1,3,8-THN  1,3,8-trihydroxynaphthalene  Tris  tris-(hydroxymethyl)-aminoethane  UV  utraviolet  vol  volume  W  white  xii  w/v  weight by volume  YM  mixed culture of yeast mycelia  xiii  Acknowledgements  It is a great pleasure to acknowledge my supervisor, Dr. Colette Breuil for her advice, and encouragement throughout my Ph.D. program from the beginning to the end. I would like to express my appreciation to my advisory committee members Dr. Jim Kronstad for helpful discussions and the use of his lab facilities for doing Southern blot in this study, and Dr. Jack Saddler for his advice and suggestions. I like to thank Dr. Y . Kubo at Kyoto Prefectural University, Japan for his gifts of cucumber seed and Colletotrichum lagenarium strains, Dr. M . Farman, at University of Kentucky, U S A and Dr. B. Valent at DuPont Agricultural Products, U S A for their gifts of Magnaporthe grisea strains, Dr. W. Hintz at University of Victoria, Canada for his gift of p A N 7 - l , Dr. L. Glass at University of California, Berkeley, U S A for her gift of pCB1004, Dr. J. Bohlmann at Biotechnology Lab, U B C for the use of his lab to perform Southern blot, and Dr. L. Stein at Cold Spring Harbor Laboratory, U S A for his comments on my fungal melanin gene system software. Appreciation is expressed to the former and the present members of the Chair of Forest Products Biotechnology for their assistance, sharing ideas and techniques, and valuable discussions. Special gratitude is extended to Brad Hoffman for the contribution of his time with patience and kindness critical reading of this thesis, Dr. S. K i m for stimulating discussions and critical reading a part of this thesis, and Dr. N. Sudetjo for reading a chapter of the thesis. Thanks are also extended to U B C Faculty of Graduate Studies for providing University Graduate Fellowships and a St. John Award, to Merck Genome Research Institute, Burroughs Wellcome Fund and Canadian Genetic Diseases Network for providing bioinformatics and genomics training fellowships. Sincere appreciation is given to my parents, brother, sister for their encouragement and perseverance. I am deeply thankful to my wife, Joanne for her endless love, patience, invaluable assistance and understanding.  xiv  Chapter 1  Introduction: fungal melanin and the DHN melanin biosynthetic pathway  Melanins, generally described as dark-brown to black pigments, are biological macromolecules composed of various types of phenolic or indolic monomers, usually complex ed with protein, or carbohydrates (Butler and Day, 1998). These pigments are produced by a variety of bacteria, fungi, protozoans, plants and animals. Because of their insolubility, melanins are difficult to characterize bio-chemically, however, utraviolet (UV), visible, infrared (IR) and nuclear magnetic resonance (NMR) spectrographic data are available for melanin from a few species (Bell and Wheeler, 1986). In this chapter, the different types of fungal melanins, the function of melanin in sapstaining  fungi,  and the  biochemical  and molecular  aspects  of  the  dihydroxynaphthalene (DHN) melanin biosynthetic pathway are described.  1.1 Fungal melanin types  Melanins are found in many fungi and can be synthesized by a variety of metabolic pathways. The types of melanins produced vary from organism to organism. Four types of  fungal  melanins  have  been  reported  based  on  their  monomers:  dihydroxyphenylalanine (DOPA) melanin, glutaminyl-3,4-dihydroxybenzene (GDHB) melanin, catechol melanin and dihydroxynaphthalene (DHN) melanin.  1  1.1.1 Dihydroxyphenylalanine (DOPA) melanin  Tyrosine is the starting monomer of D O P A melanin. D O P A is formed by the oxidation of tyrosine by a tyrosinase, as shown in Figure 1.1 (Mason, 1948). D O P A can then be oxidized to form melanin by many different enzymes, such as laccases, polyphenol oxidases, peroxidases and catalases that are found in the cell wall. Animal melanin is normally D O P A melanin, but D O P A melanin is also produced by yeasts, mushrooms and other fungi, such as Cryptococcus neoformans, Neurospora crassa and Aspergillus nidulans  (Wang and Casadevall, 1994; Prota, 1998; Brown and Salvo, 1994). The  tyrosinases used to produce D O P A in these fungi have been characterized (Horowitz and Shen, 1952; Bull and Carter, 1973), and found to contain a single copper ion necessary to bind the phenol group of the substrate.  1.1.2 Glutaminyl-3,4-dihydroxybenzene (GDHB) melanin  Glutaminyl-3,4-dihydroxybenzene (GDHB) melanin is synthesized from y-glutaminyl4-hydroxybenzene (GHB), via the shikimate pathway shown in Figure 1.2 (Rast et al., 1980). In this pathway shikimic acid can form GHB, which is then converted to GDHB, which undergoes a non-enzymatic polymerization to melanin. G H B has been found in fungal mycelia as well as in fruiting bodies, G D H B , on the other hand, has been isolated from the cell wall of Agaricus bisporus basidiospores (Rast et al., 1980). Agaricus produces a tyrosinase that oxidizes G H B to melanin, which is restricted to the  2  1,3,6,8-THN  scytalone  /  1,3,8-THN  Acetate  DHN 1,1 - D i m e r  Figure 1.1.  DOPA melanin biosynthesis (Bell and Wheeler, 1986).  3  y-N-C-CHj-CHj-CH-COOH  I 8  HO. ^ HO-v^  peroxidase /  g a.  phenolase  *• g  -  S  "  °-  = g » 2  O  v  y-N-C-CH,-CH,-CH-COOH NH, peroxidase/ phenolase  0=<  /V-N-C-CH.-CH-COOH  non- enzymatic polymerization  —  HO -MELANIN - • — protein— SH  Figure 1.2.  HO-^ ^-NH,  \=/  ^  GDBH melanin biosynthesis (Bell and Wheeler, 1986).  reproductive hyphae that form melanized spores. G H B and G D H B have been found in several species of basidiomycetous fungi, but not in ascomycetous fungi, suggesting that G D H B melanins are produced only in the basidiomycetous fungi (Wheeler, 1983).  4  1.1.3 Catechol melanin  Catechol melanin lacks nitrogen and its polymerization proceeds through free radicals or quinone-catechol adducts (Figure 1.3), however, the biosynthetic origin of catechol is unknown. The analysis of Ustilago maydis teliospore melanin found that catechol is the precursor used to produce melanin in these structures (Piatelli et al., 1965; Banuett and Herskowitz, 1996).  1.1.4 Dihydroxynaphthalene (DHN) melanin  D H N melanin biosynthesis starts with the polymerization of acetate by a polyketide synthase (PKS) to form a pentaketide (Figure 1.4). 1,3,6,8-tetrahydroxy-naphthalene (1,3,6,8-THN) is then formed by the cyclization of the pentaketide. A reductase then converts 1,3,6,8-THN to scytalone, which is dehydrated by a scytalone dehydratase (SD) to form 1,3,8-trihydroxynaphthalene  (1,3,8-THN). This compound is then  converted to 1,8-dihydroxynaphthalene (1,8-DHN) after an additional reduction and dehydration step. Finally, the oxidative polymerization of 1,8-DHN produces D H N melanin (Bell and Wheeler, 1986; Butler and Day, 1998). Although many fungi produce D H N melanin, only the melanin of a few species has been intensively studied, including Magnaporthe  grisea, Colletotrichum  lagenarium, Exophiala  dermatitidis), and Aspergillus fumigatus  dermatitidis  (Butler and Day, 1998).  5  (Wangiella  MELANIN  HO.  i  HO'  HO.  Catechol  HO' HO. HO'  Figure 1.3.  Catechol melanin biosynthesis (Bell and Wheeler, 1986).  o Pentaketide  p ^ — ^ C H  Synthase Acetyl CoA  OH  o Pentaketide Synthase  3  Pentaketide  OH Reductase HO"  0 H  P  i  .!  0"  o  ^OH  Scytalone  1,3,6,8-THN  O  OH  OH OH  ,  HO" 1,3,8-THN  OH OH Dehydratase  Reductase  ^  Scytalone Dehydratase  *"  /*v x^s v  (OO  *" MELANIN  HO' DHN  Vermelone  Figure 1.4.  DHN melanin biosynthesis (Bell and Wheeler, 1986).  6  1.2 Molecular genetics of DHN melanin biosynthesis Biochemical studies of the D H N pathway are difficult, because the intermediates of the pathway are unstable. Enzyme assays have to be performed under argon or nitrogen gas (Tajima et a l , 1989; Vidal-Cros et al., 1994). Therefore, molecular approaches have been favored to study the pathway. Isolation and characterization of D H N melanin genes are necessary to conduct molecular analysis of the pathway. Several strategies have been successfully utilized in the cloning of D H N melanin biosynthetic genes.  1.2.1 Gene cloning strategies The genetic complementation of melanin deficient mutants seems to be the most straightforward method to clone melanin synthesis genes. This method requires a wellestablished transformation system for the organism in which the complementation is to be performed. A s well, a melanin-deficient mutant of the organism in which the complementation is to be performed has to be created. For example, in order to clone a SD gene in A. fumigatus, a melanin-deficient mutant with reddish pink conidia was first created and characterized. A genomic cosmid library of A. fumigatus was then used to transform the mutant. Cosmid-restored mutants producing bluish green conidia were then rescued from the complemented strains. Finally, the SD gene arpl was mapped in one of the complementing cosmids (Tsai et al., 1997). This approach has been  7  successful in the isolation of genes encoding the PKS, reductase and SD in A. fumigatus and Alternaria alternata (Kimura and Tsuge, 1993; Tsai et al., 1997, 1998, 1999).  The isolation of melanin genes using the reverse genetics cloning approaches relies on purified enzymes. Where purification is possible, production of antibodies to the enzyme, and isolation of the gene from a c D N A expression library can be accomplished. This procedure was used to clone a reductase gene from M. grisea (Vidal-Cros et al., 1994).  Melanin genes can also be isolated by PCR-based cloning approaches. For example, the P K S gene from Nodulisporium sp. and a reductase from O. floccosum have been cloned using this approach (Fulton et al., 1999; Eagen, 1999). For example, to clone the Nodulisporium  pksl  gene, degenerate P C R primers were developed based on the  conserved regions of the C. lagenarium PKS1 gene and its Aspergillus homolog. These primers were used to amplify and clone a fragment of the Nodulisporium pksl gene. A cosmid library of Nodulisporium genomic D N A was probed with this gene fragment, and a full-length copy ofpksl was identified and cloned (Fulton et al., 1999).  Finally, melanin genes can be retrieved using cross-species hybridization. Once a gene has been cloned it can often be used as a probe for isolation of a cognate gene from a heterologous organism. The SCD1 gene encoding a SD in C. lagenarium was cloned using this approach (Kubo et al., 1996). In this case the melanin-induced c D N A library of C. lagenarium was screened using a SD c D N A from M. grisea as a probe. Then to  8  retrieve the genomic copy of the gene, a cosmid library of C. lagenarium was screened using the cloned cDNA as a probe. Similarly, the THR1 gene, encoding a reductase in C. lagenarium has been cloned using the heterologous probe, BRM2, a reductase gene involved in melanin biosynthesis in A alternata (Perpetua et al., 1996).  1.2.2 Characterization and functions of the cloned D H N melanin synthesis genes and their products  Molecular studies of melanin biosynthesis have been mainly applied to three fungal species, C. lagenarium, A. fumigatus and M. grisea. In order to assign the functions of the isolated melanin genes in these species two gene transfer techniques were used. Firstly, genetic complementation was used, and secondly, gene disruption where 'knock-out' strains are developed was used. To date, molecular studies have identified 7 different genes involved in the DHN-melanin biosynthetic pathway in fungi: a pentaketide/pentaketide synthesis gene, two reductase genes, a scytalone dehydratase gene, an aygl gene, an oxidase gene and a laccase gene.  1.2.2.1 Synthesis of 1,3,6,8-THN  The synthesis of 1,3,6,8-THN via a pentaketide intermediate has been confirmed using 2  H-,  13  C - , and C-labelled acetate feeding experiments (McGovern and Bentley, 1975; 14  Seto and Yonehara, 1977). A PKS participates in the formation of the naphthalene unit in D H N melanin by joining and cyclizing five acetate carbon units to form 1,3,6,8-THN.  9  The PKS1 gene, encoding a PKS in C. lagenarium, has been cloned, and found to contain one open reading frame (ORF) encoding a polypeptide consisting of 2187 amino acids. This protein contains a P-ketoacyl synthase (KS), an acetyl/malonyl transferase (AT), and two acyl carrier (ACP) domains (Mayorga and Timberlake, 1992; Takano et al., 1995). This PKS1 gene was over-expressed in the heterologous species, Aspergillus oryzae. Then the synthesis of 1,3,6,8-THN was successfully confirmed using in vitro cell-free extracts from C. lagenarium with C-labeled acetyl Co A and/or C-labeled malonyl 14  14  CoA as substrates (Fujii et al., 1999, 2000). These results unambiguously identified malonyl C o A as the starter as well as the extender units in the formation of 1,3,6,8-THN by the C. lagenarium PKS1. However, other fungi may still use acetyl C o A (Tsai et al., 2001). Disrupted PKS1 mutants do not synthesize melanin, but providing these mutants with scytalone can restore melanization.  Two functional homologues of the C. lagenarium PKS1, A. fumigatus albl and Nodulisporium sp. pksl (Tsai et al., 1998; Fulton et al., 1999), have been isolated. Nodulisporium pksl encodes a putative protein of 2,159 amino acids. Inactivation of this gene resulted in melanin deficient transformants. The A. fumigatus albl gene encodes a 7-kb transcript and a putative protein of 2,146 amino acids. A n albl disruptant lost its ability to produce 1,3,6,8-THN and its virulence to humans.  The amino acid sequences of Nodulisporium pksl product and C. lagenarium P K S are highly conserved (72% similarity), while A. fumigatus PKS amino acid sequence shares only 60% similarity with C. lagenarium PKS. However, the amino acid sequence of  10  A. fumigatus P K S has a significantly higher similarity, 80%, w i t h an A. nidulans heptaketide synthase encoded by wA, indicating that A. fumigatus P K S is i n fact a heptaketide synthase. (Mayorga and Timberlake, 1992). In A. fumigatus, the synthesis of the pentaketide 1,3,6,8-THN therefore occurs v i a a chain-length shortening of the heptaketide synthesized by PKS. This chain-length shortening occurs due to the protein product of a gene called aygl, w h i c h encodes a putative protein of 406 amino acids (Tsai et al., 2001). At an early growth stage, aygl deletants produce yellow conidia that gradually turn to a green color as the culture becomes older (Tsaiet al., 1999).  Molecular analysis of bacterial polyketide biosynthesis has shown that PKSs can be divided into two types (Hopwood and Sherman, 1990; Hopwood and Khosla, 1992; Hutchinson and Fujii, 1995). Type I enzymes are large, multifunctional polypeptides, each encoding the necessary enzymatic motifs for one or more cycles of condensation, while type II enzymes consist of several single-function polypeptides associated in a complex. Fungal PKSs belong to the Type I PKS family, and are further divided into two subclasses. The W A subclass consists of PKSs involved in spore pigmentation, melanin, and aflatoxin biosynthesis. The wA protein from A. nidulans is an example of the W A subclass (Mayorga and Timberlake, 1992). The MS AS  subclass consists of 6-  methylsalicylic acid synthase (MSAS) enzymes which are found in Penicillium patulum (Beck et al., 1990) and Penicillium freri (Nicolaisen et al., 1997). M S A S catalyzes the biosynthesis of 6-methylsalicylic acid, the first intermediate in the pathway leading to the mycotoxin patulin.  11  1.2.2.2 Conversion of 1,3,6,8-THN to scytalone  The conversion of 1,3,6,8-THN to scytalone requires a tetrahydroxynaphthalene reductase (1,3,6,8-THN reductase). In A. fumigatus, the arp2 gene has been cloned. This c D N A contains an 819-nucleotide ORF encoding a putative protein of 273 amino acids. A n arp2 disruptant produces reddish pink conidia and flaviolin abundantly (Tsai et al., 1997). Scytalone, a stable intermediate and branch product of the DHN-melanin pathway, has not been detected in arp2 disruptant culture extracts. These data suggest that arp2 encodes a 1,3,6,8-THN reductase.  A functional homologue of arp2, HNR encoding a 1,3,6,8-THN reductase (4HNR) has been isolated in M. grisea (Thompson et al., 2000). The arp2 deduced protein shares 50% identity to 4HNR. The HNR gene has been expressed in E. coli and its product has been purified. The molecular mass of the purified enzyme is 28.4 kDa while the predicted molecular weight of the coding sequence is 28.6 kDa. The enzyme is NADPH-dependent and involved in converting 1,3,6,8-THN to scytalone. Unlike arp2 disruptants, HNR gene disruptants produce melanin. These results suggest that another reductase is involved in this step of the melanin synthesis pathway of M. grisea (Section 1.2.2.4).  1.2.2.3 Conversion of scytalone to 1,3,8-THN  12  A SD involved in the conversion of scytalone to 1,3,8-THN, has been detected in cellfree homogenates of Cochliobolus carbonum, and Verticillium dahliae (Wheeler, 1981). The SCD1 gene encoding a SD has been cloned in C. lagenarium, this gene contains one O R F composed of 188 codons (Kubo et al., 1996). A SCD1 disruptant forms reddish-brown colonies and accumulates scytalone. The SCD1 gene complements the SD deficient C. lagenarium  mutant 9201Y and restores the wild-type  phenotype.  A. fumigatus arpl,  the apparent homologue of C. lagenarium SCD1, has been cloned  by complementing a reddish-pink conidial mutant with a A. fumigatus genomic library (Tsai et al., 1997). This gene encodes a putative protein of 168 amino acids. Disruption of arpl results in the production of reddish pink conidia and the accumulation of scytalone and flaviolin. Accumulation of these compounds indicates that the arpl disruptant is unable to convert scytalone to 1,3,8-THN. A c D N A encoding a SD has also been cloned from M. grisea (Motoyama et al., 1998), this transcript is 740 bases long encoding a predicted protein with a mass of 23 kDa. This predicted protein shares 51% identity and 68% similarity with the predicted protein encoded by arpl, which in turn shares 58% identity and 75% similarity with the SD of C. lagenarium.  SDs has been purified and characterized in Phaeococcomyces Cochliobolus miyabeanus  sp., M. grisea and  (Butler et al., 1988; Lundqvist et al., 1993, 1994; Tajima et al.,  1989). In these enzymes the catalyzing mechanism has been found to involve substrate  13  recognition and then assisting in the enolization of the substrate and finally the dehydration of the substrate by the removal of an hydroxyl radical (Basarab et al., 1999).  1.2.2.4 Conversion of 1,3,8-THN to vermelone  The conversion of 1,3,8-THN to vermelone involves a 1,3,8-THN reductase (3HNR). In M. grisea,  a reductase gene encoding a 3HNR has been isolated from a c D N A library,  and was found to contain an 846-bp ORF. Translation of the D N A sequence gives a 282-residue amino acid sequence with a predicted molecular mass of 29.9 kDa (VidalCros et al., 1994). The encoded reductase exhibits the characteristics of the short-chain alcohol dehydrogenase family. This reductase is also NADPH-dependent, and is therefore, a class B dehydrogenase (Wheeler, 1982; Viviani et al., 1990). In the reductase-catalyzed reaction, the protonation of the substrate's carbonyl oxygen has been proposed to occur through a proton shuttling mechanism that proceeds through the hydroxyl group of Tyr-178, the 3'-hydroxyl group of N A D P H , and a crystallographic water molecule, the ultimate donor (Liao et al., 2001a). This reductase, from M. grisea, is both able to convert 1,3,6,8-THN to scytalone and convert 1,3,8-THN to vermelone (Vidal-Cros et al., 1994; Thompson et al., 2000). However, this reductase prefers 1,3,8T H N as a substrate over 1,3,6,8-THN by a factor of 4.2. A 3-D structural analysis of 1,3,8-THN and 1,3,6,8-THN in the active site of the 1,3,8-THN reductase suggests a favorable interaction of the sulfur atom of the C-terminal Met-283 with the C6 C H group of 1,3,8-THN, and an unfavorable interaction with the C6 hydroxyl group of  14  1,3,6,8-THN (Liao et al., 2001b). Met is the N-terminal residue for 1,3,8-THN reductases but not for 1,3,6,8-THN reductases.  The functional homologues of the M. grisea reductase gene, THR1, BRM2, THN1 and brnl  have been cloned from C. lagenarium, A. alternata, O. floccosum and  Cochliobolus heterostrophus, respectively (Perpetua et al., 1996; Kawamura et al., 1999; Eagen et al., 2000; Shimizu et al., 1997). THR1 contains an ORF that encodes a protein of 282 amino acids and is able to complement and restore the melanization of the C. lagenarium mutant 9141 which is unable to convert 1,3,8-THN to vermelone (Perpetua et al., 1996). Sequence analysis of the BRM2 gene finds one ORF of 801 bp, which potentially encodes a 267 amino acid protein. BRM2 can complement and restore the melanization of the A. alternate brm2 mutant, which accumulates 2-hydroxyjuglone, a shunt product of 1,3,8-THN (Kimura and Tsuge, 1993; Takano et al., 1997a; Kawamura et al., 1997). Likewise, brnl has been demonstrated to be able to convert 1,3,8-THN to vermelone. The O. floccosum THN1 gene contains one ORF and encodes for a protein of 268 amino acids. This gene has been demonstrated to complement a 1,3,8-reductase deficient mutant of M. grisea. However, none of these reductases have been demonstrated to be able to convert 1,3,6,8-THN to scytalone, unlike the 3HNR from M. grisea.  Sequence comparison demonstrated that the predicted BRM2 product is closely related to the 1,3,8-THN reductases of C. heterostrophus (Brnl 95% identity; Shimizu et al., 1997), C. lagenarium (THR1 67% identity; Perpetua et al., 1996), M. grisea (69%  15  identity; Vidal-Cros et al., 1994) and to a lesser degree O. floccosum (THN1 44% identity, Eagen et al., 2000). These reductases share about 40% identities with 1,3,6,8T H N reductases (Tsai et al., 1999; Thompson et al., 2000), but do have high homology with other biosynthetic pathway enzymes. They share 50-70%o similarities with an Aspergillus parasiticus putative ketoreductase, encoded by the verl gene, which is involved in the conversion of versicolofin A to sterigmatocystin in  aflatoxin  biosynthesis (Skory et al., 1992).  1.2.2.5 Conversion of vermelone to DHN  The conversion of vermelone to D H N involves the same SD that dehydrates scytalone to 1,3,8-THN (Butler et al., 1988). The Ser-129 mutant of the M. grisea SD causes the enzyme to favor vermelone over scytalone as a substrate (Basarab et al., 1999). The sequence of SD from A. fumigatus places an A l a in the position corresponding to the Ser-129 of M. grisea SD (Tsai et al., 1997). This change may make the A. fumigatus SD more vermelone-specific.  1.2.2.6 Polymerization of DHN to melanin  The final step of the D H N melanin pathway is the polymerization of D H N molecules to form the melanin polymer. It is believed that several enzymes could be involved in polymerizing and oxidizing D H N into melanin. In previous studies, oxidases and laccases have been proposed as the polymerizing enzymes in the pathway (Bell and  16  Wheeler, 1986). Two genes, abrl and abr2 have been sequenced in A. fumigatus. Sequence analysis reveals that abrl encodes a putative protein of 664 amino acids that possesses two multicopper oxidase signatures and has 34% identity and 43%> similarity to an iron multicopper oxidase from Candida albicans (Tsai et al., 1999). Abr2, on the other hand, encodes a protein of 587 amino acids which has 41% identity to a laccase encoded by yA of A. nidulans (Aramayo and Timberlake, 1990). The disruption of either abrl or abr2 in A. fumigatus results in an alteration of the conidial color phenotype. These results indicate that the abrl and abr2 deduced proteins are possibly involved in polymerization of D H N to form melanin.  1.2.3 Genetic organization of the DHN melanin synthesis genes  Studies to date indicate that the D H N melanin synthesis genes are clustered in some fungal species, but not in others. Gene clustering is thought to be beneficial for gene regulation and facilitate the horizontal transfer of the cluster (Keller and Hohn, 1997). D H N melanin synthesis gene clusters have been identified in A. alternata and A. fumigatus  (Kimura and Tsuge, 1993; Tsai et al., 1999). The A. alternata gene cluster  (about 30 kb) contains three genes, ALM, BRMI, and BRM2, which encode a PKS, a SD, and a 1,3,8-THN reductase, respectively (Kimura and Tsuge, 1993). The A. fumigatus  gene cluster includes the 6 genes previously described (Section 1.2.2), albl,  arpl, arp2, abrl, abr2 BRMI,  and aygl in a 19 kb genomic D N A fragment. A. alternata ALM,  and BRM2, and A. fumigatus albl, arpl and arp2, are all conserved among the  known DHN-melanin pathways in other fungi. The D H N melanin synthesis genes,  17  however, are dispersed in the genome of C. lagenarium (Kubo et al., 1996). Genetic analysis of pigmentation mutants from M. grisea has shown that the D H N melanin synthesis genes are unlinked in M. grisea, as well (Chumley and Valent, 1990).  1.3 Inhibitors of the DHN melanin biosynthetic pathway  Many metabolic inhibitors have been found or developed for the D H N melanin synthesis pathway. The PKS involved in this pathway is believed to be similar to fatty acid synthetases. Therefore, cerulenin, the fatty acid synthetase specific inhibitor, produced by Cephalosporium caerulens, has been used to test whether it can block melanin production. In these studies cerulenin was found to inhibit melanin formation in the appressoria of Pyricularia sp. (Chida and Sisler, 1987), C. lagenarium (Kubo et al., 1987) and A alternata (Hiltunen and Soderhall, 1992).  (N-phenoxypropyl)-careoxamide, and carpropamid have been developed as SD inhibitors to control rice blast disease (Jennings et al., 1999; Jordan et al., 1999; Kurahashi et al., 1999). These inhibitors may be specific to the D H N melanin pathway, because SD has no known functional counterparts in plants or animals (Jordan et al., 2000). It has been shown that carpropamid treated cultures of C.  lagenarium  accumulated scytalone (Kurahashi et al., 1997; Tsuji et al., 1997). Analyses of the interaction between a SD inhibitor, carboxamide and M. grisea SD mutants show that the His-85, His-110, Val-75, Phe-158 and Phe-162 of SD have significant influences on inhibitor binding (Jordan et al., 2000).  18  The fungicides tricyclazole and pyroquilon, used to control rice blast disease caused by fungi such as M. grisea, inhibit the reductases of the D H N melanin synthesis pathway (Woloshuk et al., 1980; Yamaguchi et al., 1982). The interaction of the inhibitors with the reductase is as follows: the hydroxyl groups of Ser-164 and Tyr-178 are hydrogen bonded to the inhibitors' carbonyl oxygen, and the phenyl ring of Tyr-223 is stacked with the inhibitors' ring system (Liao et al., 2001a). The 30-fold lower affinity of the 1,3,6,8T H N reductase for pyroquilon in contrast to that of the 1,3,8-THN reductase can be explained by unfavorable interactions between the anionic carboxyl group of the Cterminal Ile-282 of the 1,3,6,8-THN reductase and C H and C H groups of the inhibitor 2  (Liao et al., 2001b).  Numerous studies have confirmed that tricyclazole specifically inhibits the reductases of the D H N melanin synthesis pathway in various fungi such as Sclerotinia sclerotiorum,  V. dahliae,  and W. dermatitidis (Buchenauer et al., 1985; Wheeler, 1981,  1982; Wheeler and Stipanovic, 1985). Therefore, tricyclazole was suggested to be a specific inhibitor of the fungal D H N melanin synthesis pathway (Wheeler and Greenblatt, 1988). However, it has since been demonstrated to inhibit reductase activities in other polyketide synthesis pathways including the biosynthesis of the nonmelanin pigment cynodontin in Pyrenochaeta terrestris (Lazarovits et al., 1989) and aflatoxin biosynthesis in Aspergillus parasiticus (Wheeler et al., 1989). Butler and Day (1998) have listed many other similar inhibitors of the reductases in the D H N melanin synthesis pathway, such as chlobenthiazone (Wheeler et al., 1989), phthalide (Uehara et  19  al., 1995; Wheeler and Klich, 1995; Chida, 1989), 2,3,4,5,6-pentachloro-benzyl alcohol and coumarin (Woloshuk and Sisler, 1982; Inoue et al., 1984a, 1984b). These inhibitors are used in either preventing fungal diseases or in studying the fungal D H N biosynthetic pathway.  1.4 The functions of fungal melanin  Fungi produce melanin in cell walls, conidial walls and other complex fungal structures, such as perithecia, sclerotia and ascocarps. In fungi, melanins have been associated with virulence, protection from environmental stresses and fungal development (Bell and Wheeler, 1986; Butler and Day, 1998).  1.4.1 Melanin's importance to fungal virulence  Melanin is essential for the virulence of some fungi. For examples, the cucumber pathogen, C. lagenarium, and the rice blast M. grisea, lose their virulence when their melanin production is blocked (Howard and Valent, 1996; Kubo et al., 1985; Kubo and Furusawa, 1986). In melanized appressoria cell wall permeability is limited, facilitating the accumulation of glycerol thereby allowing the generation of high turgor pressures required for host tissue penetration (Money, 1997). Without these high turgor pressures, the infection peg of the appressorium cannot mechanically penetrate the underlying host tissue. When melanin production is blocked, fungal pathogens, like Colletotrichum and Magnaporthe  species, form colorless appressoria, which have a reduced ability to  20  penetrate the hosts' leaves. Similarly, disruption of the albl gene encoding PKS in A. fumigatus,  results in an albino conidia phenotype which has reduced virulence in  humans (Tsai et al., 1998). Thus, conidial melanization in A. fumigatus appears to be important in the establishment of infection.  1.4.2 Melanin protects fungi from environmental stress  Melanin is essential for protecting fungi from environmental stresses, such as desiccation, extreme temperatures, U V irradiation, hydrolytic enzymes, microbial attack, and oxidization. Microsclerotia from mutants of V. dahliae blocked in melanin production cannot survive desiccation in soil, whereas wild-type microsclerotia are able to germinate when the soil is rehydrated for about 15 days (Bell and Wheeler, 1986). Conidia of an albino strain Monilinia fructicola were destroyed by heat treatment at 40°C for 30 min, while 50% of the wild-type conidia were still alive after this treatment (Rehnstrom and Free, 1996), indicating that in this fungus melanin is playing a role in resistance to high temperatures. Similarly, the melanized hyphae of Gaeumannomyces graminis  were reported to resist higher temperatures than their non-melanized  counterparts (Frederick et al., 1999). Melanin also provides U V protection for hyphae, conidia (Gadd, 1980), and yeast cells (Marbach et al., 1984). The conidia of an A. alternata  melanin-deficient mutant lost their ability to germinate after an exposure to  U V irradiation for 120 seconds, while the germination rate in the wild type was 57% after this treatment, indicating that the non-melanized conidia were more susceptible to U V (Kawamura et al., 1999). Similar results were observed with Cladosporium pp. and  21  Oidiodendron cerealis (Zhdanova et al., 1973), and in C. neoformans yeast cells, melanin also seems to provide some protection against U V (Wang and Casadevall, 1994). Fungal melanin also provides protection against oxidizing agents. Melanized cells of A. alternata and W. dermatitidis were 1000 times more resistant to hydrogen peroxide treatment than albino cells (Jacobson et al., 1995). Melanin deficient mutants of A. nidulans and Cochliobolus sativus were highly susceptible to digestion by (3-1,3 and P-l,6-glucanase (Kuo and Alexander, 1967; Old and Robertson, 1970), indicating that melanin may play an important role in resistance to hydrolytic enzymes or microbial attack. Melanin also protects certain fungi like C. sativus against lysis in natural soils (Old and Robertson, 1970).  1.4.3 Melanin is involved in fungal development  In some fungi, melanin is involved in the development of perithecia and conidia. A n albino mutant of Ophiostoma piliferum produces immature perithecia with non-viable ascospores. However, when scytalone is added to the media, melanin production is restored, and mature perithecia can be obtained (Zimmerman et al., 1996). However, other fungal species such as C. heterostrophus can develop lightly pigmented mature perithecia without melanin (Tanaka et a l , 1991). Melanin also seems to be involved in A. alternata conidial development, as A. alternata melanin deficient mutants develop small conidia with fewer septa. The septa of the conidia in this species are melanized in the wild-type strain (Kawamura et al., 1999). In A. fumigatus the surface morphology of conidia lacking melanin is altered In this case, the unmelanized mutants produce  22  smooth conidia, and the wild type strain produces conidia with echinulate surfaces that facilitate the attachment of the spores to human tissues (Tsai et al., 1998; Jahn et al., 1997; Langfelder et al., 1998). Melanin has also been reported to facilitate invasive hyphal growth in W. dermatitidis, as wild-type strains of W. dermatitidis grow through 8% agar much faster than melanin-deficient mutants (Brush and Money, 1999).  1.5 Sapstaining fungi  Sapstaining fungi produce black or brown melanin and cause the discoloration of wood. There are three major groups of sapstain fungi. The first group includes the telomorphs and anamorphs of species from the ascomyceteous genera Ophiostoma, Ceratocystis and Ceratocystiopsis. Members of this group produce melanized mycelia and perithecia, or melanized conidia. The second major group comprises the black yeasts and includes fungi such as Hormonema dematioides and Aureobasidium pullulans. Melanin is produced in and outside the cell walls of these fungi. The third group includes molds that produce masses of conidia on wood surfaces and can also cause deep stain. A. alternata, Cladosporium sphaerospermum and C. cladospoploides are examples of this third group (Seifert, 1993). Other molds such as Penicillium sp. and Trichoderma sp. can also cause superficial discoloration on the surface of wood by producing pigmented spores (Zabel and Morrell, 1993). Our group conducted a Canada wide survey of sapstain fungi in 1997-98 and found that O. floccosum was frequently isolated across Canada. This fungus is a member of the Ophiostomatoid group (Uzunovic et al., 1999).  23  The first group of sapstaining fungi can colonize wood from wounded standing trees, during harvesting, or during final wood processing. Spores of sapstaining fungi germinate on wood and fungal hyphae then can grow radially through the ray parenchyma cells in sapwood (Ballard and Walsh, 1982). These rays provide fungi with easily assimilable substrates including soluble sugars, nitrogen and lipids (Subramanian, 1983; Gao et al., 1993; Merrill and Cowling, 1966; Terziev et al., 1993). Sapstaining fungi are unable to utilize the structural components of wood cell walls, such as cellulose and lignin. Fungi move from tracheid to tracheid via bordered pits or direct mechanical penetration of the cell wall using appressoria (Seifert, 1993). Melanin may play a role in the mechanical strengthening of both modified appressoria and mycelia to penetrate wood cell walls and the torus of bordered pits (Ballard and Walsh, 1982; Gibbs, 1993). Sapstain fungi grow best on wood at temperatures between 18 and 30°C, but serious staining also occurs on wood stored at temperatures as low as 3-8°C (Gibbs, 1993).  Sapstaining fungi produce melanin that causes cosmetic defects in wood that are costly for the wood industry. Melanized hyphae and conidiophores contribute to the appearance of the wood stain (Wheeler, 1983; Zink and Fengel, 1988). Wood discolorations or stain lead to wood product devaluation and insurance claims. Canada is the world's prime exporter of softwood lumber products and these exports contributed $36.2 billion to the Canadian trade balance (COFI, 2001). The Canadian lumber industry relies heavily on export markets and must spend millions of dollars annually to control the stain problem.  24  In Canada, lumber sapstain is controlled by either kiln drying the wood or by using antisapstain chemical treatments. Kiln drying is a process that reduces the moisture content of the wood to less than 20%. At this low moisture content fungal growth is inhibited as without water many fungal biochemical and enzymatic reactions are prevented (Zabel and Morrell, 1993). In British Columbia kiln drying is not always appropriate since some offshore markets demand green lumber, and large dimension timber is difficult to dry and may suffer drying defects. Furthermore, during ocean freighter shipments to offshore markets, lumber can re-wet and fungal growth and pigmentation can occur. Anti-stain chemicals inhibit fungal growth usually by the formation of a thin barrier that prevents the germination of fungal spores on the wood substrate (Zabel and Morrell, 1993). The chemicals are applied to the surface of lumber by dipping into tanks or by spraying. Before 1990, the polychlorophenates (PCP) were used. These chemicals were highly effective in controlling sapstain, but they were also environmentally hazardous and they have been banned in Canada and many other countries (Smith, 1991). Currently, several anti-sapstain controlling agents such as 2-(2-thiocyanomethylthio) benzothiozole (TCMM), sodium borate, and didecyl dimethyl ammonium chloride (DDAC) are being used in North America. However, they have a broad spectrum of action against organisms other than wood staining fungi and are not as effective as PCP for controlling stain. Thus, there is an urgent need for a new class of anti-fungal agents that demonstrates low environmental toxicity, known environmental fate, and high target specificity.  25  1.6 Research objectives  In order to implement methods of controlling wood stain, it is necessary to acquire detailed information regarding the mechanisms of growth and pigmentation of sapstain fungi on wood. However, specific knowledge concerning the biology, biochemistry, genetics and molecular biology of sapstaining species such as the Ophiostoma species is scarce.  Some physiological and biochemical aspects of O. floccosum 387N have been investigated (Abraham, 1995; Gao, 1996). Several fungal enzymes, which play key roles in the utilization of the nutritional resources found in wood, were identified and characterized using biochemical methods (Abraham et al., 1993; Gao and Breuil, 1995a, 1995b; Gao et al., 1993; Gao, 1996). Preliminary inhibition studies show that tricyclazole and cerulenin cause the reduction of pigment production in this fungus (Eagen, 1999). Genomic Southern analysis indicated that O. floccosum possesses sequences that are homologous to other fungal D H N melanin genes. Furthermore, a gene encoding a H N reductase has been cloned and characterized in O. floccosum (Eagen, 1999). These studies suggest that O. floccosum may utilize the D H N melanin biosynthetic pathway to produce pigment. As well, Eagen (1999) has demonstrated that nutrients affect fungal pigmentation. Further molecular studies of pigment production would allow us to understand the structures, functions, expression and regulation of the melanin genes in this fungus. This information would help researchers to either find specific targets to block pigment production or create albino mutants. For example,  26  specific chemicals could be designed to target key enzymes in melanin biosynthesis in these fungi, which could then be used to control wood stain. Albino mutants could be used in biological control of sapstaining fungi particularly at the harvest site. Since sapstaining fungi cause minimal structural damage to timber, a possible control method could be to simply prevent the growth of sapstain fungi by pre-inoculating susceptible wood with albino strains of the same species. The albino strains would then use up all of the available nutrients preventing the growth of the staining fungi. Inoculation of colorless mutants of O. piliferum on wood has been shown as a possible way to control wood stain in lab conditions (Uzunovic et al., 1999).  Since there is little information on melanin biosynthesis at a molecular level in O. floccosum  387N, the overall goal of this research project was to explore and understand  the D H N melanin biosynthesis pathway in O. floccosum using a molecular approach, as biochemical techniques were likely to be difficult due to the instability of the pathway intermediates. In order to apply molecular approaches to study the functions of the genes in this pathway a genetic transformation system for this fungus was necessary. Therefore, the first objective of this research was to develop a genetic transformation system for O. floccosum 387N. A n associated goal of this study was also to try to disrupt the reductase genes involved in melanin biosynthesis in this fungus. The results of this work are discussed in Chapter 2. SD is a key enzyme in the fungal D H N melanin pathway. Searching for sequence homology has shown that SD is only used in the fungal D H N melanin synthesis pathway. If we could isolate a SD from this fungus, we could provide more evidence that this fungus utilizes the D H N melanin pathway to  27  produce pigment. In addition, the M. grisea SD enzyme structure has been determined and several inhibitors have been developed to inhibit this enzyme. These inhibitors may be used to inhibit the SD in O. floccosum. Therefore, the second objective of this research was to isolate and characterize a SD gene in O. floccosum. The results of this work are discussed in Chapter 3. During a colleague's isolation of a reductase gene from O. floccosum, two D N A sequences, which were both reductase homologs, were isolated, however, only one was further characterized. Therefore, the third objective of this research was to characterize the second reductase gene and compare it with the first reductase gene. The associated goal of this work was to compare all fungal melanin reductases and find any association between their functions and their sequences. The results of this work are presented in Chapter 4. As we know that certain nutrients affected O. floccosum pigmentation, it was of interest to explore what happens at the transcription level to the melanin genes when the fungus is fed with different nutrients. The fourth objective of this study was therefore to determine the effect of nutrients on melanin gene expression. In order to include another important D H N melanin gene in the study, a partial PKS gene was isolated from the fungus. The results of this work are presented in Chapter 5. Finally, the conclusion and future directions of the study are discussed and presented in Chapter 6.  28  Chapter 2  Transformation of three Ophiostoma species and tentative genetic disruption of O. floccosum melanin genes  2.1 Introduction  Transformation is defined as a mechanism of genetic transfer whereby pure D N A extracted from one organism is able to induce permanent hereditary changes in the cells of a second organism, to which it is added (Finkelstein, 1991). Transformation techniques for the replacement, disruption or cloning of genes were first developed in the yeast, Saccharomyces cerevisiae. In the procedure used, yeast cells were first converted to protoplasts by enzyme treatment. After re-suspending the protoplasts in an osmotic support solution in the presence of CaCl2, the exogenous D N A was added and the transformation effected by the addition, of polyethylene glycol (PEG) (Hinnen et al., 1978). This technique is based on the idea that the transforming D N A integrates via crossover events at regions of shared homology between the incoming plasmid and the genome (Shortle et al., 1982; Rothstein, 1983). This P E G mediated transformation method has since been modified and adapted for the transformation of many species of filamentous fungi. Besides the P E G protocol, electroporation has also been employed to facilitate the uptake of foreign D N A . During electroporation, an electric pulse allows  29  fungal protoplasts, conidia, or yeast cells to absorb exogenous D N A (Van de Rhee et al., 1996; Y u et al., 1999; Chadha et al., 2000). Although this technique can be used to transform intact cells, it requires special equipment, such as a gene pulser transfection apparatus and a pulse controller  Intact cells can also be transformed by biolistic bombardment. In this procedure, fungal cells are bombarded with microprojectiles, such as tungsten particles coated with a transformation vector, allowing the exogenous D N A to integrate into the fungal chromosomes (Armaleo et al., 1990; Durand et al., 1997). This method is easy to perform, but requires a gene gun. In addition, fungal cells of Colletotrichum trifolii treated with chemicals such as lithium acetate were shown to be amendable to transformation (Dickman, 1988). This technique is simple and fast, however, the transformation frequency of this method is lower than that of both the P E G mediated transformation and the electroporation methods. More recently, a transformation protocol with Agrobacterium tumefaciens has shown promising results with a few fungal species (Winans, 1992; de Groot et al., 1998; Gouka et al., 1999). A. tumefaciens has the natural ability to transfer a segment of D N A from its Ti plasmid, known as ' T - D N A ' into plant and fungal cells so that the T-DNA integrates at random into the host's nuclear chromosomes. Several fungal species such as Aspergillus awomori, Coccidioides immitis and Agaricus bisporus have been successfully transformed by simply using a co-culture of fungal cells or spores with A. tumefaciens (de Groot et al., 1998; Gouka et al., 1999; Abuodeh et al., 2000). This method is simple, fast, and produces high transformation  30  rates. In addition, it improves the targeted integration of homologous D N A and produces transformants with a low copy number of integrated D N A .  Although many fungal transformation protocols have been developed, the transformation efficiencies of some fungi are still low depending mainly on the fungal species used and not the method used for transformation. Researchers have attempted to increase the transformation frequency of filamentous fungi by using restriction enzyme-mediated integration (Sanchez et al., 1998), where restriction enzymes are mixed with the transforming D N A and protoplasts during P E G mediated transformation. This technique has been shown to increase the transformation frequency in A. nidulans by between 20 60 fold, but the integration was random.  Among the transformation protocols, most researchers so far have used the P E G mediated transformation method to introduce foreign D N A , this protocol has also been applied to transform an Ophiostomatoid fungus Ophiostoma ulmi (Royer et al., 1991).  At the start of this thesis no system to transform any of the sapstaining fungi, including O. floccosum, had been developed. Development of such a system for sapstaining fungi would allow the examination of gene functions in more detail, for instance by enabling the construction of over-expression strains and knock-out mutants. As in this project we intended to isolate and characterise O. floccosum melanin genes to understand the functions of these genes, an efficient genetic transformation system for O. floccosum was critical. In this chapter, the development of a genetic transformation system for the  31  sapstaining fungi, Ophiostoma quercus, O. floccosum, and O. piceae with the hygromycin B resistance plasmids, p A N 7 - l and pCB1004 is presented. We also described our efforts to disrupt two melanin genes, a reductase gene (THN1; isolated by Eagen, 1999) and a SD gene (OSD1; Chapter 3 in this thesis), which have been isolated in O. floccosum 387N.  2.2 Material and methods  2.2.1 Strains and culture conditions  O. floccosum 387N, which was previously misidentified as O. piceae 387N, was obtained from the Canadian Forintek culture collection. It belongs to the class Pyrenomycetes, the order Ophiostomales, and the genus Ophiostoma. In addition, three O. piceae strains (AU1, an isolate from this lab; H2009 and H2181; Brasier and Kirk, 1993) and three O. quercus strains (AU13, an isolate from this lab; H1039 and H1042; Brasier and Kirk, 1993) were used in this study.  Stock cultures were maintained on Oxoid malt extract agar ( M E A : 33g malt extract agar and lOg technical agar/1 of distilled water). To produce yeast-like cells for protoplast production, the isolates were grown either in complete medium (CM) or in proline medium (PM) at 22 ± 1°C in rotating shakers at 230 rpm for 2-6 days (Harris and Taber, 1970; Kulkarni and Nickerson, 1981). For protoplast regeneration medium (RM), C M was supplemented with 0.6 M sucrose and the pH of the medium was adjusted to 6.1.  32  Except where noted, all chemicals and restriction enzymes were obtained from Sigma Chemical Company and Amersham Pharmacia, respectively.  2.2.2 Transformation vectors  The plasmids p A N 7 - l and pCB1004 were generously provided by William Hintz (University of Victoria, Canada) and Louise Glass (University of California at Berkeley, USA), respectively. Both plasmids contain the Escherichia coli hph gene encoding a hygromycin B phosphotransferase under the control of the Aspergillus  nidulans trpC  promoter and terminator (Punt et al., 1987; Carroll et al., 1994). Plasmid p A N 7 - l has an ampicillin resistance marker gene, while plasmid pCB1004 has a chloramphenicol resistance marker gene including a functional lacZ gene allowing blue/white screening (Carroll et al., 1994). The E. coli strain DH5ct [F' / endAX hsdRXl (r -m ) supEAA thi-X +  k  recAX gyrA  k  (Naf) relAX A (lacZYA-argF) UX69 deoR (f80 dlac A (lacZ) M l 5 ] was grown  in Luria broth (LB) at 37°C and used for bacterial transformation (Sambrook et al, 1989) and plasmid preparation. Plasmid D N A for fungal transformation was purified using a Plasmid Midi-Kit (QIAGEN) according to the manufacturer's instruction.  2.2.3 Protoplast production and separation  Protoplasts of Ophiostoma species were prepared from yeast-like cells through enzymatic digestion. Yeast-like cells were harvested from the fungal cultures by filtering through three layers of sterile cheesecloth, followed by centrifugation at 3000 rpm for 5 min. A l l  33  centrifugations in this experiment were done using a Sorvall RC24 centrifuge (Dupont) at 4°C. The cell pellet was washed twice with 40 ml sterile distilled water. The washed cells were then pre-treated for 20 min with 40 ml of 25 m M 2-mercaptoethanol, 5 m M N a E D T A at pH 8.0, then washed with 40 ml sterile distilled water, and digested for 2.5 h 2  with 8 ml of filter sterilized Novozym 234 (20 mg/ml in 1 M MgSC^). Protoplasts were separated from cell debris by centrifugation at 2000 rpm for 20 min. The supernatant containing the protoplasts was mixed with 42 ml of 0.6 M KC1 and centrifuged at 640 rpm for 20 min. Then, the protoplasts were washed with STC buffer (1 M sorbitol, 50 m M CaCl , 25 m M Tris-HCl, pH 7.5), and centrifuged at 640 rpm for 20 min. The 2  protoplasts were then resuspended in STC buffer at a final density of 10 protoplasts/ml. 8  Unless immediately used for transformation, the protoplasts were stored at -70°C with 60 pi of dimethyl sulfoxide (DMSO) and 10 pi of 2-mercaptoethanol until used for transformation.  2.2.4 Transformation  The method used was a modification of the transformation procedures described by Royer et al. (1991) and Wang et al. (1988). 1.0-1.5 x 10 protoplasts in 200 pi STC were 7  mixed with 1 pi 2-mercaptoethanol, 2.5 ml of 40 or 66% P E G 3350 solution in TC buffer (50 m M C a C l , 2.5 m M Tris-Cl, pH 7.5) and 1, 3 or 5 pg vector D N A . After 40 min 2  incubation at room temperature, 36 ml of STC buffer was added to the mixture of protoplasts and plasmid D N A , and the protoplasts were harvested by centrifugation at 640 rpm for 20 min. The harvested protoplasts were resuspended in 600 pi liquid R M .  34  After 4 h incubation at room temperature, aliquots were overlayed onto R M agar containing 200 pg/ml of hygromycin B. In all the transformation experiments, single spore isolations from positive transformants were performed to ensure single nuclear origin.  2.2.5 Disruption of a reductase and SD genes  To disrupt the reductase gene, the vector pANA476, based on the vector p A N 7 - l , was constructed by Eagan (1999). Two oligonucleotides, 2DVF1 and 2DVRI57, were designed and used in P C R reactions with pBSA4.2, a plasmid containing a full-length copy of the O.floccosumreductase gene (THN1). The forward primer, 2DVF1, was designed to add a 5' Hindlll site, and the reverse primer, 2DVRI57 was to create stop codons in all of the reading frames and to add a 3' BamRl site (Figure 2.1C, 2. ID). A product with the predicted size of 476 bp was obtained (Figure 2.1 A and B)fromP C R reactions with these primers. The PCR product obtained contained the first 157 codons of the putative N-terminal coenzyme-binding domain of THN1, the two incorporated restriction sites (Hindlll and BamHl) and stop codons. This P C R product and the plasmid pAN7-l  were digested with BamHVHindlll. The two generated BamrlllHindlll  fragments were then ligated together to form the vector pANA476 using T4 ligase (Amersham Pharmacia).  To disrupt the SD gene, the vector pBSD (Figure 2.2) was constructed by cloning a 4 kb BgUli'Hindlll p A N 7 - l fragment containing the promoter region and the hph gene into the  35  Bglll/Hindlll  site of the plasmid pBSU2 which contains a full length copy of the O.  floccosum SD gene (OSD1). The BgllVHindlll  digested pBSU2 contained a 5.5 kb O.  floccosum genomic D N A fragment including a -400 bp (5' region) of the OSD1 ORF.  THN-R  pBSA4.2  B  2DVF1  , 2DVR157 476 bp  •  XXX  476  pAN7-l  PgpdA Hindlll  BamHI  D 2DVF1 Hindlll  5' GGGGTTT AAGCTT ATG TCT CCT GCA ACT GTC AAG GAC G 3' 2DVR571 BamHI  5' GGGGTTT GGATCC ACC GCT GAG CGA CTC AGC TAC TCA TGA GG 3' * * *  Figure 2.1. The construction of the THN1 disruption vector pANA476. (A) The THN reductase gene in vector pBSA4.2 was used as template D N A with (B) disruption primers 2DVF I and 2DVRI 57. The P C R product was digested with Bamm and Hindlll and (C) ligated into the BamHUHindlll site of vector p A N 7 - l to create pANA476. (D) The sequence of disruption primers showing the restriction sites and the location of the three stop codons as indicated by the asterisks.  36  S  B  111  Hindlll  pBSU2  OSD1  Bglll  Hindlll  6.5 kb hph  gene  pBSD  5.5 kb  hph gene  Figure 2.2. The construction of the OSD1 disruption vector pBSD. The pBSU2 plasmid carrying a 6.5 kb O.floccosumgenomic D N A containing the full-length OSD1 gene was digested with HindllllBgUl, thus a 400 bp OSD1 D N A fragment was removed. To construct the pBSD, the Hindlll'Bglll p A N 7 - l fragment containing the hph gene and the expression promoter was cloned into the HindlWBglTl sites of the pBSU2.  Both disruption vectors, pANA476 and pBSD were used to transform O. floccosum 387N. The techniques for protoplasting and transformation of O.floccosumwere as described above (2.2.3 and 2.2.4). Transformants were transferred onto 48-well cell culture clusters (Coining) containing B-medium agar (Abraham, 1995) supplemented with 200 pg/ml of hygromycin and 2% (w/v) filter-sterilized mannose. The transformants were incubated at 20-23°C for 30 days to observe their colour.  2.2.6 Genomic fungal DNA isolation  Genomic D N A was obtained from yeast-like cells of fungal transformants and nontransformants by using the glass bead shaking method of Ausubel et al. (1994). D N A concentrations and purities were determined by agarose gel electrophoresis or  37  spectrophotometrically  using  a  GeneQuant D N A / R N A  Calculator  (Amersham  Pharmacia).  Fungal genomic D N A from mycelia was prepared using a drilling method (Kim et al., 1999) as follows: approximately 200 mg of mycelium was collected by centrifugation or filtration using a Nitex membrane (Tetko) in 1.5 ml microcentrifuge tubes. The mycelium was broken in an extraction buffer (300 pi of 50 m M E D T A , 50 m M Tris-HCl, 3% SDS, pH 8.5) by drilling for 3 min at 600 rpm while on ice. After drilling 150 pi of 3 M sodium acetate was added and the samples were then frozen at -20°C for 20 min. The drilled mycelial extracts were then melted, centrifuged, and the D N A precipitated from the supernatant by adding an equal volume of isopropanol. The D N A was resuspended in a  400 ml of T E buffer (10 m M Tris-HCl, pH 7.5, 1 m M EDTA), extracted twice with a mixture of phenol: chloroform: isoamyl alcohol (Amresco) and precipitated by the addition of 1 ml of EtOH (100%). The pellet was washed briefly by adding 400 pi of EtOH (70%) and dissolved in 100 pi of TE buffer.  2.2.7 Polymerase chain reaction (PCR)  PCR reactions were performed in 0.5 ml Omnitubes (Gordon Technologies) using a Hybaid TouchDown Thermal Cycler (InterScienecs). P C R reactions (50 pi) contained 100 ng of fungal genomic D N A , 40 p M of each primer, 50 p M dNTPs, I X reaction buffer (10 m M Tris-HCl, pH 8.0, 1.5 m M M g C l , 50 m M KC1), and 1 unit of Taq 2  polymerase (Appligene). Thermal cycling conditions were as follows: initial denaturation  38  (94°C, 4 min); followed by 30 cycles of denaturation (94°C, 50 sec), annealing (55°C, 50 sec) and primer extension (72°C, 50 sec); and one final cycle of primer extension (72°C, 5 min). Reaction products (15 pi) were analyzed by electrophoresis on 1.2 % agarose gels in I X Tris-acetate E D T A (TAE) buffer including 1% ethidium bromide. The D N A was visualized under U V light and documented with an Image Analyzer (IS-500 Digital Image System, Alpha Innotech Co.). To amplify the hph gene, the P C R primer pair hphF (5'-ATG C C T G A A C T C A C C G C G AC-3') and hphR (5'-CT A T T C C T T T G C C C T C G GAC-3') was synthesized based on nucleotide sequences of the 5' and 3'- end of the coding region of the hph gene.  2.2.8 Novozym-aid P C R (Novo-PCR)  For PCR screening the O. floccosum transformants, spores (10 pi X 10 spores/ml) were 8  inoculated into the wells of 96-well cell culture clusters (Corning) containing 200 - 3 0 0 pi C M in each well. The cultures were incubated 30 ~ 40 hours at 28°C without shaking. Mycelium was then transferred into a sterile eppendorf tube containing 50 pi of Novozym 234 (3 mg/ml in 0.8 M KC1, 10 m M Citric acid). After incubation for 60 min at 37°C, 150 pi of dilution buffer (10 m M Tris-HCl, pH7.5, 10 m M NaCl and 1 m M EDTA) and 20 pi of 2.5% sodium dodecyl sulfate (SDS) were added to the mixture. The mixture was then heated for 3 min at 100°C, cooled on ice for 5 min and then centrifuged at 13,000 rpm, for 5 min. 2-3 pi of the supernatant was removed for P C R as mentioned above but with the addition of 2% DMSO.  39  2.2.9 Genomic DNA dot blot and Southern hybridization analysis  hph D N A was PCR-amplified from the p A N 7 - l plasmid, separated on a 1.2% agarose gel in I X T A E buffer containing 1% ethidium bromide and purified using a GeneClean Spin Kit (BiolOl). hph D N A was then labelled with 3000 Ci/mmole [ P]oc-dATP (Amersham 32  Pharmacia) using the Random Primers D N A Labelling System (GIBCOBRL). Two pg of genomic DNAs from the transformants and non-transformants were then dot-blotted onto a Zeta-Probe GT membrane (Bio-Rad) using a BIO-RAD dot blot system as recommended by the manufacturer. The membrane was prehybridized at 65 °C for 1 hour in 20 ml of hybridization solution (7% SDS, 0.25M N a H P 0 , pH7.2). Hybridization 2  4  with the hph probe was performed overnight at 65°C in hybridization solution. The membrane was then washed twice for 30 min at 65°C first in 5% SDS, 20 m M Na2HP04, pH 7.2, and then in 1% SDS, 20 m M N a H P 0 , pH 7.2, and finally subjected to 2  4  autoradiography using Kodak X - O M A T film.  For performing Southern blot analyses, fungal genomic DNAs (10 pg each sample) were digested overnight at 37°C with Hindlll, which does not digest hph, fractionated in a 1.0% agarose gel, denatured in 0.25M HC1 for 15 min, and transferred onto Zeta-Probe GT blotting membrane (Bio-Rad) using 0.4M NaOH as a transfer solution. Southern blot analysis was performed with hph D N A as a probe. Probe labelling and hybridization conditions were the same as those described above.  2.3 Results  40  2.3.1 Transformation system  In order to transform O. floccosum, O. piceae and O. quercus, we produced protoplasts from fungal yeast-like cells (Figure 2.3). Once high quality protoplasts were obtained, the protoplasts were transformed using two different transformation vectors. Conditions for obtaining transformants, protoplast production and transformation efficiency were examined.  Figure 2.3. Yeast cells (A, X 100) and budding cells (B, X 100) and protoplasts (C, X 400 and D, X 400) of O. floccosum after treating the yeast cells with Novozym 234.  2.3.1.1 Optimization of protoplast production conditions  Several important variables such as cell age and Novozym 234 concentration were tested to determine the optimum conditions for protoplast release and regeneration in O. floccosum 387N. It was found that in cultures with  OD oo 6  values from 0.6 to 0.8, budding  cells (Figure 2.3B) were actively produced and were most sensitive to digestion by Novozym 234. The production of budding cells and protoplast yield decreased when the OD no 6  value of the cultures reached or was above an OD of 1.0 (Table 2.1).  41  Table 2.1. The relation among OD o values, yeast-like cell budding rates, and protoplasting rates of O.floccosum387N cultured in proline media. 60  OD oo value  Cell budding rate  6  (%)  Protoplasting rate  0.6  76  83  0.8  70  80  1.0  33  28  (%)  In preliminary tests, Novozym 234 concentrations of less than 10 mg/ml did not release protoplasts from O. floccosum yeast-like cells, while Novozym 234 concentrations of 30 mg/ml destroyed most cells within 1.5 h digestion. Novozym 234 concentrations of 20 mg/ml resulted in the release of large numbers of protoplasts in a 2 h digestion (Figure 2.3 C and D; Table 2.2). The obtained protoplasting rates were found to vary with the different batches of Novozym 234 used, and Lot number of 96H0503 was determined to be the most effective for protoplasting yeast cells.  Table 2.2. Production of protoplasts of O.floccosum387N using different enzyme concentrations and digestion time.  Concentration of Novozyme 234 (mg ml ) -1  10  20  Digestion time (h) 1.0 1.5 2.0 1.0 1.5 2.0  42  Protoplasting rate (%) 20 52 57 50 76 83  Fungal cells pre-treated with 25 m M 2-mercaptethanol for 20 min released 5 times more protoplasts than cells without pre-treatment. However, increasing pre-treatment time to 40 min or increasing concentrations of 2-mercaptethanol to 50 m M did not improve the protoplasting rate. With 100 ml cultures of yeast-like cells grown in P M , about 9.5 x 10  6  protoplasts was generally obtained under the optimum conditions.  The optimum conditions for protoplasting O. floccosum  387N were then applied to the  two fungal species, O. piceae (strains H2009, H2181, and AU1) and O. quercus (strains H1039, H1042, and AU13). Protoplast production from the O. piceae isolates was similar to 387N, with 70-90% of the total cells forming protoplasts. However, protoplast production from the O. quercus isolates reached only 20-30% as these isolates did not produce actively budding cells in P M . These results led us to examine the growth form of these fungi in C M .  A l l the isolates of O. floccosum,  O. piceae and O. quercus produced yeast-like cells in  C M , and it was found that yeast-like cells at OD oo values ranging from 1.4 to 1.9 were 6  most suitable for protoplasting, with protoplast production of 90-100%) within 2 h of digestion. Protoplast yields reached around 5 x 10 protoplasts from 30 ml cultures of the 7  cells. These results showed that to obtain high protoplast yields, C M is better than P M . Interestingly, there was no apparent difference in the protoplasting rates among O. floccosum,  O. piceae and O. quercus when the isolates of these three species were grown  in C M .  43  2.3.1.2 Transformation conditions and regeneration  Before performing transformation of the protoplasts, their sensitivity to hygromycin was tested. A l l the isolates were sensitive to 100 pg/ml hygromycin, except isolate H2009 which was sensitive to 150 pg/ml. Consequently, to ensure dominant selection for the hygromycin resistance marker, the regeneration medium was supplemented with 200 pg/ml hygromycin. Regeneration rates of protoplasts on R M agar without hygromycin were about 60-70% for all the isolates tested.  To examine the effects of several parameters on the transformation efficiency initial experiments were performed with O. floccosum 387N using either 3 or 5 pg of p A N 7 - l . In preliminary tests with 40% P E G , only - 2 0 transformants were obtained for each experiment. Thus, the P E G concentration used was increased up to 50% or 66%. Both concentrations produced high transformation rates displaying above 10 transformants per pg of vector D N A , however, a 30 - 90 times higher transformation rate was obtained with 66% P E G than with 50% P E G (Table 2.3). Testing with different molecular weights of P E G molecules, P E G 3350 and P E G 8000, did not have much effect on the transformation rate. The transformation rate decreased by 10% when the P E G used was pre-warmed at 37°C. Decreases in transformation rates were also observed when the protoplasts were stored overnight at 4°C or at -70°C. The transformation rate of freshly prepared protoplasts was 1.1 x 10 transformants/pg vector D N A , while the 5  transformation rates of 4°C and -70°C stored protoplasts were 7.0 x 10 and 2.7 x 10 4  transformants/pg vector D N A , respectively.  44  3  Table 2.3. The effects of PEG concentrations on transformation rates of O. floccosum 387N transformed with different amounts of pAN7-l DNA.  PEG (%) 50 66  Transformants/ pg DNA 3 pg D N A 1.3 X 10 3.7 X 10 3  4  Transformants/ pg DNA 5 pg D N A 1.2 X 10 1.1 X 10 3  5  2.3.1.3 Transformation of various O. piceae and O. quercus isolates with pAN7-l and pCB1004  The optimum transformation conditions determined for 387N were applied to the O. piceae and O. quercus isolates with both p A N 7 - l and pCB1004. Transformants were obtained from all the isolates. The effects of different concentrations of transformation vector on the transformation rates of these isolates were examined. The transformation rates were 10 times lower with 8 or 10 pg of D N A than with 1, 3, or 5 pg of D N A from either plasmid. Therefore, all the isolates were transformed with 1, 3, or 5 pg of either pAN7-l or pCB1004. Three rounds of transformation were performed and the results are summarized in Table 2.4.  For all the isolates tested, the transformation rates were about 10 ~10 transformants/pg 4  5  D N A for either p A N 7 - l or pCB1004. For O. quercus, all three isolates yielded high transformation rates when 1 pg of either plasmid was used in the transformation. A l l of the O. floccosum and O. piceae isolates yielded the highest transformation rates when 1 pg of the pCB1004 D N A was used.  45  Table 2.4. The effects of DNA amount on the transformation rates of O. floccosum, O.piceae and O. quercus isolates transformed with pAN7-l or pCB1004. Transformatants/ pg DNA  Transformatants/ pg DNA Isolates  387N H2009 H2181 AU1 HI 042 H1039 AU13  pCB1004(pg)  pAN7-l (pg) 1 1.3 x 10 8.2 x 10 5.2 x 10 3.3 x 10 4.8 x 10 4.0 x 10 1.2 x 10  3  5  4  4  5  5  5  3.7 3.7 2.0 5.0 1.6 2.4 4.8  3 x x x x x x x  10 10 10 10 10 10 10  4  5  4  4  5  5  4  5 1.1 x 10 1.9 x 10 9.0 x 10 2.4 x 10 1.5 x 10 8.8 x 10 5.3 x 10  5  5  4  4  5  4  4  6.0 7.7 7.5 3.3 4.7 5.7 1.0  1 x 10 x 10 x 10 x 10 x 10 x 10 x 10  4  5  4  4  5  5  5  1.2 2.6 2.3 3.4 2.5 1.6 5.8  3 x x x x x x x  10 10 10 10 10 IO 10  3  5  4  4  5  5  4  5 1.2 x 2.0 x 3.6 x 1.5 x 1.9 x 8.5 x 3.9 x  IO IO 10 10 10 10 IO  3  5  4  4  5  4  4  Notes: 387N: O. floccosum 387N strain; H2009, H2181 and A U 1 : O. piceae strains; H1042, H1039 and AU13: O. quercus strains.  2.3.1.4 Analysis of transformants  Transformants from each isolate with either p A N 7 - l or pCB1004 were observed as pinhead colonies after 2 or 3 days of incubation at 23 °C on R M agar containing 200 pg/ml of hygromycin B. Approximately two percent of the transformants grew with a normal growth rate, whereas the rest of the transformants grew slowly. Sixty transformants for each isolate of O. floccosum, O. piceae and O. quercus were randomly selected and grown on C M medium containing 300 pg/ml of hygromycin. Among these transformants, 65% were unable to grow, suggesting that only 35% of the transformants were stable. From the transformants that grew seven transformants were randomly selected for each isolate and used for molecular analysis. Genomic D N A was isolated from both the selected transformants and the wild type strains of O.floccosum,O. piceae  46  and O. quercus and subjected to P C R analysis, hph D N A was successfully amplified from all the transformants through PCR, whereas no amplification of hph D N A was observed from the wild type isolates (Figure 2.4). Dot blot analysis using a radiolabelled hph gene fragment confirmed that the transformants contained the hph gene sequences (Figure 2.5). However, differences in signal intensity in the dot blot hybridization among the transformed isolates were observed.  pAN7.| 3 4  5 6  pCB1004  7 8 9 10 11 12 13 I- 15 16  Figure 2.4. P C R analysis of Ophiostoma isolates transformed with plasmids pAN7-l and pCB1004 using primers specific for hygromycin B phosphotransferase gene.  PCR products (10 pi) were separated electrophoretically in a 1.2 % agarose gel and stained with ethidium bromide. Lanes 1 and 8: AU13; 2 and 9: H1042; 3 and 10: H1039; 4 and 11: A U 1 ; 5 and 12: H2181; 6 and 13: H2009; 7 and 14: 387N; 15: wild type 387N; 16: lkb D N A ladder. Arrow indicates location of hygromycin B phosphotransferase D N A bands. 387N: O. floccosum 387N strain; H2009, H2181 and A U 1 : O. piceae strains; H1042, H1039 and AU13: O. quercus strains.  47  To evaluate the mitotic stability of the transformants, the seven randomly selected transformants from each isolate were transferred six times onto C M agar at 5-day intervals and transferred back onto C M agar containing 300 pg/ml and 800 pg/ml hygromycin B. A l l transformants retained resistance to hygromycin, indicating good mitotic stability.  I I fym  2 t  3  •  4 |  m £(b  5  6  I I -  7  C  I  - PAN7-I  *  -DCBI004  Figure 2.5. Dot blot analysis of Ophiostoma isolates transformed with plasmids p A N 7 - l and pCB1004. Genomic D N A (2 pg) of each isolate was blotted on Zeta-Probe membrane and hybridized with P-dATP-labeled hygromycin B phosphotransferase DNA. Lanes 1: AU13; 2: H1042; 3: H1039; 4: A U 1 ; 5: H2181; 6: H2009; 7: 387N; C: wild type 387N. 387N: O. floccosum 387N strain; H2009, H2181 and A U 1 : O. piceae strains; H1042, H1039 and AU13: O. quercus strains. 32  2.3.2 Genetic disruption of melanin genes in O. floccosum  After establishing the transformation system, we attempted to disrupt a reductase and a SD gene in O. floccosum. The objective of this work was to elucidate the functions of these genes in O. floccosum melanin biosynthesis.  2.3.2.1 Genetic disruption of an O. floccosum 387N reductase gene  48  To disrupt the reductase gene, O. floccosum 387N was transformed using the reductase gene disruption vector, pAN-A467. Three rounds of transformation were performed using pAN-A467 D N A , and about 150 transformants were retrieved from C M agar containing 200 pg/ml hygromycin. Most of the transformants were brownish after 3 weeks of incubation. Around 10% of the transformants were dark-brown in colour.  To find any reductase gene disruptants we screened the transformants using a P C R approach. The primer combinations of 414F/hphF, and 414F/1756R (414F: 5' C C T TTC G G A CTT C A G A A T G C 3' and 1756R: 5' A T T GTC C G A GGT G G C A A T A A C G 3') were used to screen the transformants (Figure 2.6). In the first round of screening, five light-brown and 11 dark-brown O. floccosum transformants were screened. With the primer combination of 414F/hphF, no PCR product was amplified in these transformants, but PCR products of about 1.4 kb were amplified with the primer pair of 414F/1756R (Figure 2.7). This size of P C R product was the same as that of the P C R product amplified from the wild type strain with the same primers. This suggested that no homologous integration had occurred in these transformants. Due to the difficulty of visually determining the colour of these fungi, we further PCR-screened the remaining O. floccosum transformants. However, no reductase gene disruptant was found.  49  pANA476 1  476  hph  X  genomic  • 414F  1 7 5 6 R  npnF lkb 1.8 kb  Figure 2.6. The assumed integration event by homologous recombination of the pANA476 into genomic 387N D N A . The location of PCR primers and the sizes of the predicted products if occurs as in the model.  10  9 8 7 6  5  4  3  2  1  M  Figure 2.7. P C R screening pANA476 transformed O. floccosum transformants. P C R products (15 pi) were separated electrophoretically in a 1.2 % agarose gel and stained with ethidium bromide. Lanes 1: wild type strain, 387N; lanes 2, 4, 6, 8 and 10: transformants used to perform P C R using primers 414F/1756R; lanes 3, 5, 7 and 9: transformants used to perform PCR using primers 414F/hphF; lane M , lkb D N A ladder.  50  2.3.2.2 Genetic disruption of an O. floccosum 387N SD gene  We retrieved 1392 and 808 transformants after transforming 387N with a circular or •Sacl-digested linear OSD1 disruption vector (pBSD), respectively. To observe the colour of the transformants, they were transferred onto B-medium supplemented with 2% mannose. After a 4-week incubation the transformants were divided into five groups based on their colour and growth patterns (Table 2.5). Among the 2100 transformants, two produced mixed culture of yeast mycelia with white colour (YM), while the others produced mycelia only. Twelve transformants were white (W) and one hundred and twenty eight transformants were white but changed to slightly brownish after 20-day incubation (LB). Many transformants (1050) produced a brown colour similar to the wild type strain (B), whereas 868 transformants were dark brown (DB). P C R was used to search for the integration of the target vector into the wild type OSD1 gene in the transformants. One hundred seventy transformants belonging to different phenotypic groups (2 Y M , 12 W, 20 L B , 70 B, and 66 DB) were screened using PCR. In the PCR reactions, the primer pair SD23/SD24 (SD23: 5' G G C C T C A A T A T C A G C A G C C T C A 3 ' ; SD24: 5' A A C C A G C G G A T C T T G G G A T C G A 3 ' ) was used to detect whether the whole or partial vector D N A was inserted into the transformants. Using this primer pair, a 0.6 kb fragment could be amplified from the wild type strain. At the same time, the primer pair PANS/SD24 (PANS: 5' A C T C G T C C G A G G G C A A A G G A A 3') was used to detect junctions between the recipient OSD1 and the integrated vector D N A (Figure 2.8). If integration into the genomic OSD1 gene had occurred, a P C R product of  51  1.0 kb would be amplified. However, the 170 transformants screened produced a 0.6 kb fragment with the primers SD23/SD24 and no product when the primers PANS/SD24 were used (Figure 2.9). This suggested that the OSD1 gene was not disrupted in these transformants.  Table 2.5. Different phenotypes of the transformants transformed using the OSD1 disruption vector pBSD. Phenotype White (W) Mixed culture of yeast mycelia (YM)  Number of the transformants 12 2  Light Brown (LB)  168  Brown (B)  1050  Dark brown (DB)  868  Characteristics Mycelia with white colour Mycelia and yeast-like cells with white colour Mycelia with white colour after 20-day incubation but turned to slightly brownish after 30-day incubation Mycelia with light brown colour which is similar to the wild type strain Mycelia with dark brown colour  Because the OSD1 disruptant was not found it was desirable to screen the remaining transformants (-1,940). However, screening of fungal transformants using P C R is limited by the difficulty of extracting genomic D N A . Therefore, we examined several different fungal genomic D N A extraction methods to determine which method was the most efficient. When using the drilling method, we could extract genomic D N A from 12-20 samples per day, while we could work on 40 samples per day using the glass-bead method. However, fungal yeast-like cells were required in the glass-bead D N A extraction  52  6.5 kb  Wild type  X pBSD 5.5 kb  hph gene hph gene  Mutant  5.5 kb  PANS  SD23  SD24  Figure 2.8. The assumed integration event by homologous recombination of the pBSD into genomic 387N D N A . The location of PCR primers and the sizes of the predicted products if occurs as in the model.  10  9 8 7 6 5 4 3 2 1  — 500 bp  Figure 2.9. P C R screening pBSD transformed O. floccosum transformants. P C R products (15 pi) were separated electrophoretically in a 1.2 % agarose gel and stained with ethidium bromide. Lanes 1: wild type strain, 387N; 2-6: transformants used to perform PCR using primers SD23/SD24; 7-10: transformants used to perform PCR using primers SD23/PANS; M : lkb D N A ladder.  53  method and 387N did not produce many yeast-like cells. Therefore, we developed a Novozym-aid PCR (Novo-PCR) protocol to screen the remaining fungal transformants. In this protocol, Novozym 234 is used to release the genomic D N A from the fungal cells as it removes fungal cell walls.  Several conditions were examined to improve the efficiency of the protocol. In NovoPCR reactions (20 pi), different final concentrations of Triton X-100, 0.1%, 0.5% and 1% were tested. PCR products were easily amplified when Triton X-100 was added at 0.5% or 1%>, while no PCR products were observed when Triton X-100 was added at 0.1% (Figure 2.10). Final concentrations of 2%, 5%, 8% and 10% of D M S O were also tested in the PCR reactions. A strong band was observed when the reaction contained 2% D M S O (Figure 2.11). This protocol allowed us to screen 100 samples per day. Therefore, we screened -1,940 transformants using Novo-PCR and the primers mentioned above.  9  8 7 6 5 4  3 2 1 M  Figure 2.10. Novo-PCR with Triton-100. PCR products (15 pi) were separated electrophoretically in a 1.2 % agarose gel and stained with ethidium bromide. Lanes 1: wild type strain, 387N; 2-9: pBSD transformed O. floccosum transformants used to carry on PCR with primers SD23/SD24. Triton-100 was added in the P C R reaction up to 1% (lanes 5, 9), 0.5% (lanes 1, 2, 4, 6, 7) and 0.1% (lanes 3, 8 respectively).  54  9  8 7 6 5 4  3 2 1 M  Figure 2.11. Novo-PCR with D M S O . PCR products (15 pi) were separated electrophoretically in a 1.2 % agarose gel and stained with ethidium bromide. Lanes 1: wild type strain, 387N; 2-9: pBSD transformed O. floccosum transformants used to carry on PCR with primers SD23/SD24. D M S O was added in the PCR reaction up to 2% (lanes 1, 6-8), 5% (lane 2), 8% (lane 3) and 10% (lanes 4-5).  Figure 2.12. Southern hybridization of the O.floccosumtransformants with pBSD. Genomic D N A (10 pg) of each isolate was blotted on Zeta-Probe membrane and hybridized with P-dATP-labeled hph D N A . Lanes 1: 387N and 2-7: transformants. 32  Unfortunately, no OSD1 gene disruptant was found by PCR-screening the 1,940 transformants. To confirm the integration of the disruption vector into the fungal genome,  55  20 transformants were selected to perform Southern blot analysis using hph D N A as a probe. The wild type 387N strain was included as a negative control. No band was visible in the control, but 2-8 bands of at least 12 kb were seen in the transformants (Figure 2.12).  2.4 Discussion  2.4.1 Transformation  In the transformation study, Novozym 234, a multi-enzyme extract of Trichoderma was used as it has been shown to be effective in digesting the fungal cell walls of many fungi (Beach & Nurse, 1981; Brygoo & Debuchy, 1985; Ballance et al., 1983, 1985; Yelton et al., 1984; Vollmer & Yanofsky, 1986). This enzyme preparation contains a complex mixture of hydrolytic enzymes, with high levels of 1,3-glucanases and chitinases. Novozym 234 concentrations of less than 10 mg/ml, which worked in other filamentous fungi, did not release protoplasts well in 387N yeast-like cells. A Novozym 234 concentration of 20 mg/ml produced the highest protoplasting rates with the Ophiostoma isolates tested. It is known that the Ophiostoma species have cellulose in addition to chitin in their cell wall (Jewell, 1974). In addition, chitin contents in fungal cell wall are affected by culture media, culture conditions and cell age (Ouellette et al., 1995). These findings might explain why a higher concentration of enzyme was necessary to obtain protoplasts in our studies.  56  We also found that different batches of Novozym 234 affected the protoplasting rates of our isolates, similar findings have been reported by other researchers (Akins & Lambowitz, 1985; Kinnaird et al., 1982; Kinsey & Rambosek, 1984). This is probably due to the lack of specificity of the enzyme preparation, since these preparations are not marketed specifically for fungal protoplasting. It was found that P E G concentrations influenced the transformation rate. Higher concentrations of P E G increased the transformation efficiency, as high concentrations of P E G likely cause the treated protoplasts to clump, and this may facilitate the trapping of D N A (Fincham, 1989). We observed that the incubation of P E G , D N A and protoplasts at 37°C decreased the transformation efficiency. This may be due to protoplast damage occurring at these temperatures as it has been reported that temperatures above 30°C damage protoplasts (Shirahama et al., 1981). Transformations of freshly made protoplasts were more efficient than transformations of protoplasts that were kept overnight at 4°C or 1 month at -70°C. These data suggested that low temperatures influenced the stability of 387N protoplasts, subsequently reducing the transformation rate. As well, different vectors and their amounts affected the transformation rates of the isolates. When we used pCB1004 to perform the transformations, the transformation rates were higher at lower pCB1004 concentrations. These results were in agreement with the data reported for A. nidulans transformation (Yelton et al., 1984).  In the D N A dot blot analysis, differences in signal intensity of dot hybridization were apparent among the transformed isolates, suggesting that different numbers of hph gene copies were present in the transformants. The Southern blot analysis of the transformants  57  confirmed that several copies of the transformation vector were integrated into the fungal genome. It seems that these fungi are good recipients of foreign D N A , and this could explain why the transformation rates of our isolates were 100 to 1000 times higher than the transformation rates in other fungi, which reach value of about 1 0 - 1 0 transformants per pg of D N A (Fincham, 1989).  2.4.2 Gene disruption  Disruption of the melanin genes THN1 and OSD1 from 387N, was attempted using the established transformation system. The transformants were screened using a P C R approach but no disruptants were found. The attempts to disrupt THN1 in 387N by a colleague also failed (Eagan, 1999). In her attempts, five putative THN1 disruptants were obtained by P C R screening. However, hybridization studies of these putative disruptants suggested that targeted integration of the disruption vector into the genomic gene had not occurred and that the PCR results were artefacts.  Our attempts to disrupt the THN1 and OSD1 genes and Eagen's attempts to disrupt the THN1 gene suggested that homologous recombination in O. floccosum might be a rare event, although the transformation efficiency of O. floccosum was high. Homologous recombination is also a rare event in Cryptococcus neoformans, Histoplasma capsulatum and Coccidioides immitis transformants (Lodge et al., 1994; Woods and Heinecke, 1996; Y u and Cole, 1998). Phenotypic characterization of the transformants, however, showed that some of the transformants were significantly different from the wild type strain, as  58  some of the transformants had reduced or increased pigmentation. A possible explanation for these differences may be that the transformation event, while not appearing to affect the OSD1 or THN1 genes directly, may have randomly integrated into other genes, which may be involved in the regulation of melanin biosynthesis. Our Southern blot analyses of the transformants showed that multiple copies of hph were integrated into the O. floccosum  genome. Heterologous D N A  transformation  of  species  from  integration  Trichoderma,  has been reported  Gliocladium,  in  the  Cryptococcus  and  Coccidioides (Lorito et al., 1993; Toffaletti et al., 1993; Y u and Cole, 1998). Furthermore, the relative level of non-homologous integration versus integration at a site of homology in filamentous fungi depends on both the gene being utilized and the specific recipient strain that is employed (Kim and Marzluf, 1988).  Recently, a gene was successfully disrupted in another Ophiostoma species, O. ulmi (Hintz, 1999). The transformation rate of O. ulmi is similar to that of our Ophiostoma isolates. In their work, the disruption cassette was constructed so that the target gene flanked a dominant selectable marker on both sides. This type of disruption vector may facilitate homologous integration. In our attempts, we tried to construct a similar disruption vector but we could not find the appropriate enzyme digestion sites to place the dominant selectable marker in the middle ofOSDl.  In conclusion, a transformation system has been established in several Ophiostoma fungi including O. floccosum. The optimum conditions for protoplasting and transformation, such as cell age and Novozym 234 and P E G concentrations have been determined. This  59  information will help other researchers to perform transformation of sapstaining fungi. The attempts to disrupt two melanin genes in O. floccosum have failed. Molecular analyses of the transformants in our experiments and others suggest that homologous recombination in O. floccosum may be a rare event.  60  Chapter 3  Isolation and characterization of an O. floccosum OSD1 gene that complements a Colletotrichum lagenarium melanin-deficient mutant  3.1 Introduction  SD is one of the key enzymes in the D H N melanin biosynthetic pathway. This enzyme converts scytalone into 1,3,8-THN and is probably also involved in dehydrating vermelone into 1,8-DHN (Butler and Day, 1998). It was the first enzyme of the fungal DHN-melanin pathway to be isolated and characterized, from both Phaeococcomyces sp. and C. miyabeanus (Butler et al., 1988; Tajima et al., 1989). The studies of the crystal structure of M. grisea SD show that the enzyme has a novel folding pattern that buries the hydrophobic active site in its interior when the substrate is bound (Lundqvist et al., 1993, 1994). A detailed mechanism of catalysis for the M. grisea SD was proposed following site-directed mutagenesis of its active-site residues (Zheng and Bruice, 1998; Basarab et al., 1999). Active site residues Tyr-30, Asp-31, Tyr-50, His-85, His-110, Ser-129, and Asn-131 were all found to be important in substrate binding and catalysis. Ser-129 participates in the orientation of the substrate within the active site, while Asn-131 is involved in positioning the substrate for binding and in the protonation of the substrates' carbonyl through donation of hydrogen to its' phenolic oxygen atom. Tyr-30 and Tyr-50 assist the protonation of the substrates' carbonyl through the water molecule. His-85 provides a general base and a general acid in the reaction. Asp-31 and His-85 form a  61  dyad that increases the basicity of the His-85 imidazole. His-110 is thought to have a role in stretching the C3 carbon-oxygen bond by sharing a hydrogen bond with the hydroxyl group.  Furthermore, M. grisea SD has been used as a molecular target for designing inhibitors to block disease-causing fungal melanin production (Jennings et al., 1999; Basarab et al., 1999; Jordan et al., 1999). Several inhibitors such as amids and carpropamid have been developed. SD has no known functional counterparts in plants or animals, therefore inhibitors for this enzyme should decrease the risk of deleterious effects occurring in offtarget organisms (Jordan et al., 2000). These inhibitors can block fungal melanin production and thus, protect plants from fungal diseases caused by melanin producing pathogens.  The SD genes of C. lagenarium (Kubo et al., 1996), M. grisea and A. fumigatus (Tsai et al., 1997) have been isolated and characterized. Both C. lagenarium and M. grisea are phytopathogenic fungi and cause diseases on cucumber and rice, respectively. A. fumigatus is a human pathogen which causes invasive pulmonary aspergillosis (Tsai et al., 1997). Disruption of C. lagenarium SD gene blocks the conversion of scytalone to 1,3,8-THN in the melanin biosynthetic pathway. Scytalone accumulates in these disruptants which produce a reddish pigment on potato sucrose agar (PSA), form nonmelanized appressoria on glass slides, and do not have the ability to infect cucumber leaves (Kubo et al., 1996). Likewise, disruption o f M grisea and A. fumigatus SD genes results in the disruptants accumulating scytalone and producing reddish pigment (Tsai et  62  al., 1997; Butler and Day, 1998; Thompson et al., 2000). As mentioned in Section 1.4.1 melanized appressoria are essential for host penetration and thus these disruptants are not able to penetrate host tissues (Kubo et al., 1985, 1996).  In this chapter, we present the isolation of the OSD1, a SD gene of O. floccosum, and the complementation of a SD deficient mutant of a distantly related pathogenic fungus, C. lagenarium  using the cloned O. floccosum SD gene.  3.2 Materials and methods  3.2.1 Fungal and bacterial strains, culture media, vectors and PCR primers  C. lagenarium wild type strain 104-T and mutant 9201Y were provided by Dr. Kubo at Kyoto Prefectural University, Japan. The fungal strains were grown on potato dextrose agar (PDA; Difco). Culture storage procedures were the same as those described previously (Section 2.2.1). Mycelia of C. lagenarium were produced from two cores of C. lagenarium  macerated in potato dextrose broth (PS) using a blender. Cultures were  incubated in a rotary shaker at 20°C for 7 days. Escherichia coli strains DH5cc (Life Technologies) and LE392 (Promega) were used for the propagation of plasmids and bacteriophage lambda E M B L 3 (Promega) clones, respectively.  The plasmid pBSKII+ (Stratagene) was used for general D N A manipulation. The plasmid, pBSU2 was produced by subcloning a 6.5 kb BamHI fragment of O. floccosum  63  genomic D N A containing the SD gene into pBSKII+. The transformation vector, pESD was used for complementing the C. lagenarium SD deficient mutant 9201Y. This vector was constructed by ligating the 6.5 kb BamHI genomic D N A fragment containing the SD gene into BamHI digested p A N 7 - l , which contains a hygromycin-resistance hph gene as a selective marker. PCR and D N A sequencing primers are listed in Table 3.1  3.2.2 Nucleic acid manipulations and genomic DNA library amplification Bacterial plasmid DNAs were purified using the Plasmid Midi Kit (QIAGEN) according to the manufacturer's instruction. Phage D N A was isolated using the Q I A G E N E lambda D N A isolation kit. For the genomic D N A extraction from C. lagenarium and O. floccosum  and  the drilling method described in Section 2.2.6 was used. Restriction digestions  ligations  were  performed  according to manufactures'  instructions  using  commercially supplied enzymes and buffers. Mycelia harvested and frozen at -80°C were used for R N A extraction. Total R N A was extracted using the RNeasy Plant Extraction kit (QIAGEN) after drilling the frozen mycelia at 600 rpm for 3 min on ice or smashing liquid nitrogen-frozen conidial or yeast cells.  A n O. floccosum 387N genomic D N A library was constructed by Eagen (1999) using the E M B L 3 replacement vector (Promega) and the manufacturers' protocol. The library was amplified as follows: an aliquot of the library containing 10 plaque forming unit (pfu)/ml 5  phage was used to infect the host bacteria L E 392. The mixture was plated on a 150 mm L B plate, and incubated for 8 hrs at 37°C. The amplified library was titered(10 pfu/ml) u  and stored at -80°C.  64  Table 3.1. List of the oligonucleotides synthesized for use in sequencing and as PCR primers. Use  Remark  Name  Sequence  SD1  5' G A G T G G GCI GA(T/C) (T/A) (C/G)IT A(C/T) G A 3'  PCR  F  SD2  5' CCI GC(G/A) AA(C/T) TTC CAI A C I C C 3'  PCR  R  SD5  5' G A T T C C A A G G A C T G G G A C CGT C 3'  PCR  F  SD6  5* CGT C G A CCT TGC GGT A C C A G T G 3'  PCR  R  SD3  5' CTC A C A C A A GTT G C C GTC A A G 3'  Sequencing  F  SD10  5' C T G TCT GTT A G C A A G A A G A T C 3'  Sequencing  R  M13F  5' TGT A A A A C G A C G G C C A G T 3'  Sequencing  F  M13R  5' C A G G A A A C A GCT A T G A C C 3'  Sequencing  R  SD23  5' G G C C T C A A T A T C A G C A G C CTC A 3'  PCR  F  SD24  5' A A C C A G C G G A T C T T G G G A T C G A 3'  PCR  R  SD26  5' T G C G C C G A T G C A A T G G C A CT 3'  PCR  R  Note: F represents forward primer, while R represents reverse primer  3.2.3 DNA hybridization using fluorescein-labelled probes  D N A fragments were labelled with fluorescein using the Gene Images Random Prime Labelling System (Amersham Pharmacia) according to the manufacturers' instructions. The labelling reaction (50 pi) used 150 ng of probe D N A and was incubated for 4 hours at 37°C. The reaction was terminated by adding E D T A to a final concentration of 20 mM. The D N A transferred membranes were hybridized with 100 ng of the fluoresceinlabelled D N A overnight at 60°C in 0.1% SDS/5% SSC (0.3 M N a citrate, 3 M 3  65  NaCl)/5%(w/v) dextran sulphate/20 fold dilution of the block solution (Amersham Pharmacia). The membranes were first washed in 0.1% SDS/1% SSC for 15 min at 60°C, and then in 0.1% SDS/0.5% SSC for 15 min at 60°C. Signal detection was performed using a Gene Images Detection Kit (Amersham Pharmacia).  3.2.4 PCR-based cloning of the OSD1 gene  Several conserved regions of the SD amino acid sequences of C. lagenarium (GenBank accession number: D86079) and M. grisea (GenBank accession number: AB004741) were used to create SD degenerate primers. The regions chosen were based on the codon usage weight and preference. A codon frequency table (based on several genes from various filamentous fungi) was used to help determine which nucleotide to substitute in the third codon position. Furthermore, the primers were evaluated to avoid any direct repeats, internal hairpin structures or dimerization. Two degenerate oligonucleotides, SD1 and SD2, were designed. SD1 is fully degenerate to the conserved amino-acid sequence, E W A D S Y D , and SD2 is fully degenerate to the conserved amino-acid sequence, G V W K F A G (Figure 3.1).  SD1 and SD2 were used as primers with genomic D N A from 387N in order to amplify SD sequences from this fungus. Reaction products (30 pi) were analyzed on 1.2% agarose gels. The desired PCR-amplified D N A fragments were excised, and purified using the GeneClean kit (Bio 101). The purified P C R amplified products were ligated into pBSKH+ using T4 ligase and then transformed into competent DF£5oc E. coli cells  66  C1SD  PAGN  MgSD  A.EEFI A.EEFV  C1SD MgSD  59 62  LFEWADSYDSKDWDRLRKffllAPg L R I D Y R S F L D K I W E A M P /YEWADSYDSKDWDRLRKmlAPi LRIDYRSFLDKLKIEAMP EDJADSYD  FIGGSRWEKVS D T R FIGGTRWEKVS EDRJ  C1SD  :  MgSD  :  3H AH S HNM HKJ Y R K VN G W K F AGLi SH AH S Hw L HW Y K K I D G W K F AGL GWKFAG  C1SD  :  FAAAH  MgSD  :  HQLRVPHQKY HQLRVPHQRY K BTHMKISBTJ  1  121 124  STEDQKTDVKWEKEIK  :  183  JDK  :  172  : 188 :  Figure 3.1. A n amino acid sequence alignment of the SDs of C. lagenarium and Af. grisea. Conserved regions are black shaded and locations of the degenerate oligonucleotides are indicated by the arrows. Dashes indicate absent amino acids necessary for alignment purposes.  using the calcium chloride procedure described by Sambrook et al. (1989). Transformed colonies were screened using white/blue selection based on the insertional inactivation of (3-galactosidase. Five white colonies were selected from LB/amp/Xgal plates for plasmid D N A preparations. Plasmids were checked by P C R using the primers SD1 and SD2 to ensure that the appropriate insert was present. DNAs were purified from the confirmed clones and sequenced.  To clone the full-length gene, an obtained fragment, showing high sequence homology to fungal SDs, was used as a probe to screen an  O. floccosum  genomic D N A library. For  screening the library, an aliquot of the amplified library was used to infect L E 392 bacteria and plated on 150 mm L B plates at approximately 10 pfu/plate. Plaques were 5  transferred onto a Hybond-FU- nylon membrane (Amersham Pharmacia). The membranes were then hybridized with the fluorescein-labelled PCR fragment as described above. Six  67  positive plaques were picked up and the recombinant lambda phage D N A s purified and digested with restriction enzymes. The digested DNAs were fractionated in a 1.0% agarose gel, and transferred onto a Hybond-H+ membrane. Southern blot analysis was performed with the fluorescein-labelled probe. The identified D N A fragment containing the putative OSD1 was cloned into pBSKJJ+ and sequenced.  3.2.5 Complementation of C. lagenarium mutant 9201Y  Mycelia of 2-day old C. lagenarium mutant 9201Y were grown in liquid culture (100 ml) for 2 days then harvested by filtration using a Nitex membrane (Tetko) and treated with Novozym 234. The procedures of protoplast production and transformation were the same as those described in Section 2.2.3. Approximately, l x l O lagenarium  7  protoplasts of C.  mutant 9201Y were transformed with 20 pg of the transformation vector  pESD containing the full length OSD1 gene sequence. Transformants were selected on P D A plates with 1 M glucose and 200 mg/ml of hygromycin. Single spore isolations were then performed to ensure single nuclear origin.  3.2.6 RT-PCR analysis of OSD1 expression in transformants The first strand of cDNAs was synthesized using 6 pg of DNase treated total R N A , a Oligo(dT) primer and A M V Reverse Transcriptase (Promega) according to the manufacturer's instruction. PCR conditions were the same as described in Section 2.2.7. Detection of the OSD1 transcript from the first strand of cDNAs was performed by PCR using the gene-specific primers, SD23 and SD26, which were designed based on the  68  0SD1 D N A sequence. A 0.6 kb fragment would be amplified i f OSD1 was expressed in the transformants. RT-PCR was conducted on c D N A from wild-type strain, the Scd" mutant 9201Y, the obtained melanin-restored transformants of C. lagenarium, and O. floccosum 387N as a positive control. R N A samples from O. floccosum not treated with reverse transcriptase were included as a negative control. The P C R products were resolved on a 1.2 % agarose gel by electrophoresis.  3.2.7 Pathogenicity test of transformants  Inoculation and pathogenecity assays on cucumber leaves were performed as described by Kubo et al., 1982. Cucumber (Cucumis sativus L.; "Suyo") seeds were kindly provided by Dr. Y . Kubo. Cucumber leaves were excised from one-month old plants. Conidia were collected in water by brushing culture plates of C. lagenarium wild-type strain, mutant 9201Y and 4 melanin-restored transformants. The obtained conidial suspensions were centrifuged at 2,000 g for 2 min. 20 pi of resuspended conidia (10  5  conidia/ml) was spotted on the surface of the cucumber leaves. After inoculation, the leaves were incubated in humid petri dishes at 24°C for a week before observation.  3.2.8 DNA sequencing and computer analysis of sequence data  D N A sequencing was performed at the Nucleic Acid Protein Service of University of British Columbia. AmpliTag FS DyeDeoxy™ terminator (Applied Biosystems) was used for the sequencing. Sequencing primers are listed in Table 3.1.  69  PCR primer design was performed using the P C R primer design program, GeneFisher (http://bibiserv.techfak.uni-bielefeld.de/genefisger/). Database searches were performed using the B L A S T server at the National Centre of Biotechnology Information (NCBI; Altschul et al., 1990). D N A and protein sequences were used as to search the nr or est databases at the N C B I web server. The parameters used in the searches were as follows: BLOSUM-62 matrix, ungapped search, E (Expected)-value of 1.0 and masking was turned on. The ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html),  and GenScan (Burge and  Karlin, 1997) programs were used for protein translation and ORF and intron prediction. PepTool 2.0 (BioTool) was used to predict protein chemical and physical properties. The P C - G E N E software package (IntelliGenetics) and clustalW (Thompson et al., 1994) server at European Bioinformatics Institute and GeneDoc program (Nicholas & Nicholas, 1997) were used to conduct the protein sequence alignment and analysis. Phylograms were constructed from alignments using the P A U P * 4.0b6 for Windows/DOS (Sinauer Association Inc). Multiple sequence alignments were converted into distance matrixes and then converted into trees using the neighbor-joining method of clustering. The resulting trees were displayed using TreeView (Page, 1996). A bootstrapped protein distance analysis was also performed on the same sequence alignments to generate 1000 resampled datasets and CONSENSE was used to construct a consensus tree (Brinkman and Leipe, 2001). The search for potential transcription factor binding sites was performed with the matrix search program Matlnspector (Quandt et a l , 1995) using the TRANSFAC  4.0  matrices  (Wingender  70  et  al.,  2000).  ProtComp  nittp://www.softberryxoiTi/protein.htm  was used to predict the probability of the  subcellular localization of proteins, and has a prediction accuracy of 70-90%.  3.3 Results  3.3.1 Isolation and characterization of a putative SD gene  3.3.1.1 Cloning of the OSD1 gene  In order to isolate a SD D N A sequence from 387N, a PCR-based cloning method was used. The degenerate primers, SD1 and SD2 were designed based on two highly conserved regions of amino acid alignment between the SDs of C. lagenarium and M. grisea. In the P C R reactions with 387N genomic D N A and the primers SD1 and SD2 only one amplicon of approximately 420 bp was produced (Figure 3.2, lane 1). No amplification occurred in the control reactions (Figure 3.2, lanes 2, 3). The PCRamplified D N A fragment was subcloned and five transformants were tested to check whether they contained the 420 bp insert. P C R using SD1 and SD2 as primers showed that the 420 bp band was present in all the transformants (Figure 3.3, lanes 1-5). One of the positive clones was designated as OCD and chosen for sequencing.  A B L A S T X search of the OCD sequence in the N C B I nr database found 2 hits, one with a C. lagenarium and the other with M. grisea SD amino acid sequences, with significant E value (le" ). The deduced protein sequence (Figure 3.4) from O C D shared 83% 50  identity and 91% similarity to the C. lagenarium SD amino acid sequence, and  71  1  1 kb  —  500 bp  —  M  2  3  Figure 3.2. P C R products of genomic D N A of O. floccosum using SD1/SD2 (lanes 1), SD1 (lanes 2) and SD2 (lanes 3) as primers. The lane labeled ' M ' is the one kilobase ladder (BRL).  5  4  3  2  1 M  — 1.6 kb  _ —500 bp  Figure 3.3. P C R products amplified with plasmid D N A from bacterial transformants with the primer combinations of SD1/SD2. The lane labeled ' M ' is the one kilobase ladder (BRL).  72  77% identity and 88% similarity with M. grisea sequence. These results suggested that the 420 bp O C D sequence was a fragment of the SD homolog.  Before screening the library, we checked whether the library contained the putative SD gene by PCR. One, two, five, ten and twenty pi of aliquots of the library were used as templates in the P C R reactions. The nested primers SD5 and SD6, designed based on OCD sequence information were used in the reactions. A specific band of 400 bp was observed in each sample (Figure 3.5), indicating that the putative SD gene was in the library. The library was screened by colony blot hybridization using the fluoresceinlabelled 420 bp O C D sequence. Six positive plaques were found. Crude phage plugs were picked up, subdivided, and amplified in E. coli LE392 to perform another round of screening. Finally, three putative clones were recovered.  The D N A of one of the positive clones was analyzed by restriction digestion with BamHI, Hindlll,  HindllllSad,  Rsal,  and EcoRI, respectively (Figure 3.6A). Digested  DNAs were transferred onto a Hybond N+ membrane and a Southern blot analysis was performed using fluorescein-labelled OCD sequence as a probe. A 6.5 kb BamHI fragment containing the putative SD gene (Figure 3.6B) was then subcloned into the BamHI  site of pBSKII+ to produce pBSU2 (Figure 3.7). The plasmid was checked by  digestion with BamHI to ensure that a fragment of the appropriate size was present. The BamHI  fragment from the plasmid was sequenced using the specific primers (SD3 and  SD10) designed based on the OCD sequence information.  73  1  gagtgggccgacagctatgattccaaggactgggaccgtctgcgc E W A D S Y D S K D W D R L R  46  aagtgcattgctcccactctgcgaatcGTACGTCTTATACAACCC K C I A P T L R I  91  TGCTTTCTCTATTAACCCATAAAGATCTTCTTTGCTAACAGACAG  136  gactaccgctcgttcctgaacaagctgtgggaggccatgccggcc D Y R S F L N K L W E A M P A  181  gaagagtttatcggcatgatctccgaccccagcgttctcggcaac E E F I G M I S D P S V L G N  226  cccctgctgcgcacacaacacttcttcggcgcctcgcgctgggag P L L R T Q H F F G A S R W E  2 71  cgcatctccgacaccgaggtcgtgggctaccatcagctgcgcgtc R I S D T E V V G Y H Q L R V  316  ccccaccaggtctacacagataccactctcacacaagttgccgtc P H Q V Y T D T T L T Q V A V  3 61  aagggccacgcccactcggccaacacccactggtaccgcaaggtc K G H A H S A N T H W Y R K V  4 06  gacggcgtctggaag D G V WK  Figure 3.4. DNA sequences and deducted amino acid sequence of the 420-bp PCR product. The D N A sequence is shown in the upper strand, and the amino acid sequence is shown in the lower strand. Intron is depicted in italic letters.  74  4  5  3  2  1  M  Figure 3 . 5 . P C R products amplified with 3 8 7 N genomic l i b r a r y aliquots (lanes 1-5: 2 0 p i , 1 p i , 5 p i , 2 pi and 1 0 pi,) with the nested primer combinations S D 5 / S D 6 .  4  5  5  3  2  3  4  1  2  M  1  Figure 3 . 6 . Identification of the D N A fragment containing OSD1. (A) Gel patterns of  the positive phage D N A digested with Hindlll (lane 1), BamRl (lane 2), HindWSacl (lane 3), Rsal (lane 4) and EcoRl (lane 5), and (B) identical blots hybridized with O CD fragment.  75  A  B  Figure 3.7. The construction of the vector pBSU2. (A) The 6.5 kb BamHI fragment of a genomic X phage clone showing the orientation and approximate location of the OSD1 gene, (B) The cloning vector pBSKJJ+ used to accommodate the fragment.  3.3.1.2 Nucleotide sequence and deduced amino acid sequence of O.floccosumOSD1  A total of 1477 nucleotides designated as OSD1 (GenBank accession Number: AF316575) was obtained by sequencing pBSU2. OSD1 contained 315 bp upstream sequence of the translation initiation codon (ATG), an ORF with 648 bp, and 372 bp downstream sequence of the stop codon (TAG) (Figure 3.8). A short sequence characteristic of a potential Hogness box ( T A T A A A A ) was observed. The first A T G occurred 140 nucleotides downstream of the Hogness box and was the most likely translation initiation codon. Sequences sharing significant homology with several fungal transcription factor binding sites such as A B A A , NIT-2, and HAP234 were detected upstream from the putative start codon. Among these elements, the nitrogen regulatory binding site NIT-2 protein (Fu and Marzluf, 1990) was present at 3 locations in the 5' upstream region (Figure 3.8). A potential polyadenylation signal, A A T A A , was present  76  112 bp downstream from the stop codon. However, the significance of these detected sites remains to be elucidated. The G C content of the coding sequence was 61%. Two introns were found in OSD1; one of 76 bp and the other of 63 bp in length were located at the nucleotide positions 79-154 and 257-319, respectively. The organization of introns and exons in OSD1 started with a 78 nucleotide exon followed by a 76 nucleotide intron, then a 102 nucleotide exon followed by a 63 intron and a 478 nucleotide exon.  The putative spliced OSD1 sequence encodes a predicted polypeptide sequence of 216 amino acids (Figure 3.8). The predicted molecular weight and theoretical isoelectric point of the deduced OSD1 protein were 24.2 kDa and 6.07, respectively. A l a , Asp and Ser appeared as the three major amino acids comprising the protein sequence. The frequencies of these amino acids in the protein sequence were ~ 2% higher than their frequencies in an average protein, while the frequencies of Asn, Cys and Gly were ~ 2% lower than their frequencies in an average protein (Table 3.2). The percentage of hydrophilic amino acids comprising the sequence was 51.4% and the ratio of hydrophilicity to hydrophobicity (HR) of the sequence was 1.3. Protein solubility (S) was 1.56. The OSD1 deduced protein was predicted to locate in the cytoplasm.  77  315 27 0 225 180 13 5 - 90 -4 5 1 1  TGGTCTATTGTTAGTAGCTATATGTATTTCT7AATGATAGGCATAT GGCATACTGAGTTACTTCTTTTTTTGATTGTCTTGCTTACTTGGC ACGTCTCGACAAGGTGTGACCAGACTCTCGGGCTCCTCCATGGGA AGGCCGTAGTGTCTGAGACCAGACTCTTGATATAAAAGTAAAGCT GTGACGATCTAGTTTCTAGATTTCATCTCATTCAAAAGTTCTTCT TTTCTTCCTTTTTTCGACGATCGAGGACAAGCTGACTAGTATCGA CAAGACACATTTTTAACGGATTCATACTCTCACCAACCAGCACAA atgggcctcaatatcagcagcctcacatcgaccacgtcgagcgcg M G L N T S S L T S T T S S A  46 1 6 91  cccaagacaaccggcagcgacatctcctttgagGGTATTTCCTAT P K T T G S D I S F E TCTCCTCAAAATGCTCGCCAAATAAAAAAACAAAACAAGAAAACA  13 6 27  AGAAGGACACTAATAGCTAgactacatgggcctctgcagtgccgc D Y M G L C S A A  181 36  ctacgagtgggccgacagctatgattccaaggactgggaccgtct Y E W A D S Y D S K D W D R L  226 51  gcgcaagtgcattgctcccactctgcgaatcGTACGTCTTATACA R K C I A P T L R I  2 71  ACCCTGCTTTCTCTATTAACCCATAAAGATCTTCTT.TGCTAACAG  316 61  ACAGgactaccgctcgttcctgaacaagctgtgggaggccatgcc D Y R S F L N K L W E A M P  3 61 75  ggccgaagagtttatcggcatgatctccgaccccagcgttctcgg A E E F I G M I S D P S V L G  4 06 c a a c c c c c t g c t g c g c a c a c a a c a c t t c t t c g g c g c c t c g c g c t g 90 N P L L R T Q H F F G A S R W 451 105  ggagcgcatctccgacaccgaggtcgtgggctaccatcagctgcg E R I S D T E V V G Y H Q L R  4 96 120  cgtcccccaccaggtctacacagataccactctcacacaagttgc V P H Q V Y T D T T L T Q V A  541 135  cgtcaagggccacgcccactcggccaacacccactggtaccgcaa V K G H A H S A N T H W Y R K  586  ggtcgacggcgtctggaagtttgccggcctcgatcccaagatccg  78  150  V  D  G  V  W  K  F  A  G  L  D  P  K  I  R  631 165  ctggttcgaatacgattttgataaggtgtttgccagcggccgcga W F E Y D F D K V F A S G R D  676 180  ccagtttggcaccgaggagaaggcggcagcaactgccggaccaga Q F G T E E K A A A T A G P E  721 195  actcctcgccaaggacaaggtgcagagtgccattgcatcggcgca L L A K D K V Q S A I A S A Q  766 210  gagagccgtggccgtcagtgcttagTTTGTCTATTGTAATTTAGT R A V A V S A STOP  811 TTGTTGTTTTCTTTTGCTAAACGCGTATGGGAGCTGTTTTGTTCG 856 ACACCTTCTCAAGTCTACTTGTCCATCTTATTTATTGCACTTGAA 9 01 AGAATAACCAGGCCGTTTACCTTGTTGTATGAATAGAGTAATCAC 94 6 ATTGAGCACATTGAGCGCACTTGAGAAAGACTTTAGGAAAAATCA 991 AATTCTAAATGAAATACATATCGGTATCTGAGCAGTAATTGGGTA 103 6 ATCCGTAAGGCCAAACGCAAAAAAGACAAAGCAGACAGACATAGA 1081 AGCAAAGAACCTAAACTGGAAACAACAGAGAACAGAAAACGTTAA 112 6 AAAGTAAAAGTGAATGAAAAGAAACGAAATGTCATGC  Figure 3.8.  Nucleotide and deduced  amino acid sequences  of OSD1  f r o m O.  floccosum. Numbers in the left margin indicate position relative to the nucleotide of the start codon, or amino acid position. The start codon (atg) and stop codon (tag) are bolded. Upper case nucleotides in the coding region indicate the introns. The potential Hogness box is bolded and underlined. The putative binding site of transcription factor nit2 in the 5' untranslated leader sequence is underlined. The potential polyadenylation signal sequence is in italic and bolded.  79  Table 3.2. Inferred amino acid composition of O. floccosum OSD1 deduced protein.  Amino acid Ala (A) Arg(R) Asn (N) Asp (D) Cys(C) Gin (Q) Glu (E) Gly(G) His (H) He (I) Leu (L) Lys(K) Met (M) Phe (F) Pro (P) Ser (S) Thr(T) Trp (W) Tyr(Y) Val (V)  No. 24 12 4 16 2 .7 11 13 6 9 15 12 4 10 8 20 15 7 8 13  Real frequency (%) 11.1 5.6 1.9 7.4 0.9 3.2 5.1 6.0 2.8 4.2 6.9 5.6 1.9 4.6 3.7 9.3 6.9 3.2 3.7 6.0  Expected freq 8.8 4.2 4.6 5.9 2.1 3.7 5.9 8.3 2.1 5.4 8.0 6.3 2.0 3.7 4.5 6.6 6.0 1.4 3.7 7.1  3.3.1.3 Comparison of the OSD1 deduced protein sequence with other fungal SDs  A B L A S T X scan of the N C B I nr database found 3 hits: SDs of C. lagenarium with an Evalue at 1 0 , M. grisea with an E-value at I O -74  51  -69  and A. fumigatus with an E-value at 10~  (GenBank accession number: AAC49843). OSD1 deduced protein sequence showed a  high degree of identity and similarity with the SDs of C. lagenarium (69% and 87%), M. grisea  (70% and 89%) and A. fumigatus (51% and 78%). The alignment of these  sequences revealed the presence of four regions that were conserved in all of the fungal SD sequences (Figure 3.9).  80  MGLNISSLTSTTSSAPgTTGSDg  SJYEWAD S Y D S K D W D R L R K H I A P  MG  JVJYEWAD S Y D S K D W D R L R K H I A P  SQVQ@SD--E MASPAG--Ni  I I J F E W A D S Y D S : K DW D R L F . K H I A P  MVEJaKPNLTg  {JFBCTADSYDB|KDWDRLRMIAP  PAEEFI PAEEFV PAEEFI PAEBYM  Of SD MgSD ClSD Af SD  Of SD MgSD ClSD Af SD  p K IRS IF E Y  :  168  E D IMS G E F  :  15 6  E E IRK S E Y  :  153  ET  :  155  T E E KAAAT AGPE LIZARD!VQSAIASAQRAVAVSA  EY  216 172  Q | T DVKWE KEI KF AAAH  TED  188 168  Figure 3.9. Comparison of the deduced amino acid sequence of O. floccosum OSD1 (OFSD) with those of C. lagenarium (CLSD), Af. grisea (MGSD), and A. fumigatus (AFSD). Dashes indicate gaps introduced to maintain the alignment. Perfectly conserved and well-conserved positions in the alignment are shaded with black and grey colours respectively. The residues comprising the active site are indicated as *.  The predicted OSD1 amino acid sequence showed a C-terminal extension and a N terminal extension. The active site residues Tyr-30, Asp-31, Tyr-50, His-85, His-110, Ser-129, and Asn-131 in M. grisea SD, and C. lagenarium SD as Tyr-27, Asp-28, Tyr47, His-82, His-107, Ser-126, and Asn-128 were well conserved in the O. floccosum OSD1 deduced protein as Tyr-42, Asp-43, Tyr-62, His-97, His-122, Ser-141, and Asn143 (Figure 3.9).  31  3.3.2 Functional characterization of OSD1 gene  3.3.2.1 Complementation of the C. lagenarium mutant 9201Y with O. floccosum OSD1  As the OSD1 disruption failed, the functional analysis of the gene product was conducted by genetic complementation. The vector pESD (Figure 3.10) was used to transform the C. lagenarium  mutant 9201Y. The plasmid p A N 7 - l was included as a control for the  transformation. Two transformation experiments were conducted using 10 pg of the vectors in each transformation. Transformants were selected from P D A plates containing 200 pg/ml hygromycin. Fifty five hygromycin-resistant transformants were retrieved and transferred onto 24-well cell culture cluster (Corning) plates containing P D A agar and 600 pg/ml hygromycin for further characterization.  3.3.2.2 Characterization of C. lagenarium transformants  The colour of the transformants was used as an indication that complementation had occurred. Approximately, 24 out of 52 hygromycin-resistant transformants produced a greenish-black color on P D A plates. The restored color was slightly lighter than that of wild type strain 104-T (Figure 3.11).  82  BamHI  \  AMP  BamHI  * BamHI pAN7-l  B  HPH  Figure 3.10. The construction of the transformation vector, pESD. The BamHI fragment (A) from pBSU which was inserted into the BamHI site of pAN7-l (B). H P H : hygromycin phosphotransferase gene, AMP: ampicillin resistance gene.  104-T  9201Y  Transformant  Figure 3.11. The colour of C. lagenarium cultures. 104-T: wild type, 9201Y: Scd" mutant, and transformant: a melanin-restored O. floccosum OSD1 gene transformant. The integration of OSD1 into the genome of the melanin-restored transformants was examined by Southern blot analysis. Genomic D N A , extracted from wild type strain 104-  83  T, mutant 9201Y, 5 melanin-restored transformants of C. lagenarium and O. floccosum 378N, was digested with EcoRl. The blots were hybridised with the full length OSD1 sequence. There is no EcoRI site in OSD1. A l l the transformants displayed at least one band in the blot. Five bands were observed in one of the transformants (Figure 3.12). No band was detected in either C. lagenarium wild-type strain or mutant 9201Y. A single band of OSD1 gene was detected in O. floccosum 378N (Figure 3.12).  m  * '  *""* * 5  r  F i g u r e 3.12. G e n o m i c S o u t h e r n b l o t a n a l y s i s o f t h e C. lagenarium S c d m u t a n t t r a n s f o r m e d w i t h O. floccosum OSDlgene c a r r y i n g p l a s m i d p E S D . Lanel: O. floccosum, lanes2-3: C. lagenarium wild type strain and Scd" mutant 9201Y, lanes 4-7: melanin-restored transformants.  The expression of OSD1 in the melanin-restored transformants was examined. RT-PCR was performed using total R N A extracted from the mycelia of the C. lagenarium wild type strain, mutant 9201Y, 4 melanin restored transformants and O. floccosum 387N. The primer pair, SD23/SD26, which is specific for the O. floccosum OSD1 sequence, was used in the P C R reactions. A n expected fragment of 0.6 kb was produced in the four transformants and O. floccosum, but not in the mutant 9201Y and the wild type strain of C. lagenarium (Figure 3.13). No signal was detected in R N A samples without the treatment with reverse transcriptase.  84  8  7  6  5  4  3  2  1 3 Kb  — 1Kb  Figure 3.13. RT-PCR detection of the O.floccosumOSD1 gene transcript from the C. lagenarium Scd" mutant transformed with the O.floccosumOSD1 gene. Lane M : 1 kb D N A ladder, lane 1: O. floccosum, lanes 2-6: melanin-restored transformants, lane 7: Scd" mutant 9201Y, lane8: wild rype strain of C. lagenarium.  The pathogenicity of the C. lagenarium melanin-restored transformants was examined. Conidia of C. lagenarium wild type strain, mutant 9201Y and 4 melanin-restored transformants were inoculated on cucumber leaves. After 7-day incubation, the wild-type strain and the melanin restored transformants caused necrotic lesions on the cucumber leaves. In contrast, the mutant 9201Y did not cause any necrotic lesions on the cucumber leaves (Figure 3.14).  Figure 3.14. Pathogenicity test of the melanin-restored transformant complemented with O. floccosum OSD1 gene. Two cucumber leaves were inoculated with the transformant, mutant 9201Y, and wild type strain 104-T of C. lagenarium. Mutant 9201Y could not form necrotic lesion (spots 3 , 4, 6), wild type strain 104-T (spot 5) and the melanin-restored transformant (spots 1, 2) form necrotic lesions.  85  3.4. Discussion 3.4.1 Characterization of OSD1 gene  A n OSD1 gene from O. floccosum 387N was isolated by a PCR-based cloning approach. Database searches using this gene sequence retrieved three fungal SDs that had very low B L A S T E-values between 10" and 10" . E-values below 10" indicate that the query 51  71  50  sequence is a match to the target in the database and i f the alignment covers the whole of both proteins, then there is a good chance that they share the same or a related function (Brenner et al., 1998). These results suggested that the OSD1 is a structural gene encoding a SD. This conclusion was confirmed by the complementation of a C. lagenarium SD deficient mutant using OSD1.  The number and position of introns in OSD1 and SD genes from C. lagenarium and A. fumigatus were similar. The organisation of the SD gene in M. grisea is not known because only c D N A sequence encoding the SD is available. In OSD1 two introns of 76 bp and 63 bp were identified. In C. lagenarium the introns were 57 bp and 67 bp while in A. fumigatus the introns varied from 49 to 54 nucleotides. The size of these introns was in agreement with the concept that fungal introns have an average length of less than 100 bp (Gurr et al., 1987). The first and third exons in OSD1 were larger than those of SD genes from C. lagenarium, M. grisea and A. fumigatus. Thus OSD1 encoded the longest SD protein reported so far, with 216 amino acids. The signal sequences in the putative intron matched  the  consensus  sequences  of  86  the  N.  crassa 5'  splice  signal  GGT(A/G/T)(A/C/T)G(T/C), and the 3' splice signal (A/T)(T/C)AG (Bruchez et al., 1993), suggesting that introns in this gene conformed to filamentous fungal consensus splice sequences.  The OSD1 protein's predicted molecular weight was similar to that of the purified SD (23 kDa) of C. miyabeanus (Tajima et al., 1989). The percentage (51.4%) of hydrophilic amino acids of the OSD1 deduced protein is higher than the average percentage of hydrophilic amino acids (47.6%) of a naturally occurring soluble protein, suggesting it is hydrophilic. This is consistent with the fact that M. grisea SD is a hydrophilic protein (Lundqvist et al., 1993, 1994). It is reported that highly hydrophilic enzymes have lower thermal stability, for example, lipase at 50°C loses 75 % activity (Longo and Combes, 1997). This suggests that high temperatures may decrease the activity of or inactivate the SD in this fungus and may thereby block melanin production.  The predicted SD protein from O. floccosum was more closely related to the M. grisea SD than the C. lagenarium and A. fumigatus SDs. It is important to notice that active site residues of M. grisea SD are well conserved in the O. floccosum OSD1 deduced protein and C. lagenarium SD, suggesting that the OSD1 deduced protein has the same catalytic function as the M. grisea and C. lagenarium SDs.  The study of inhibitor binding interactions with the M. grisea SD showed that Val-75, His-85, His-110, Asn-131, Phe-158, Phe-162 are major amino acid residues that contribute to binding of the inhibitor (Jordan et al., 2000). These amino acids were well  87  conserved in the OSD1 deduced amino acid sequence (Figure 3.9; marked by @). Therefore, inhibitors of the M. grisea SD may inhibit the O. floccosum SD and reduce pigmentation, due to the high sequence similarity between the two enzymes. In fact Fleet (2001) applied the M. grisea SD inhibitor carpropamid to O. floccosum and found that the inhibitor did reduce the melanization of this fungus. The ProtComp program predicted that the OSD1 protein was located in the cytoplasm, as well the locations of the other three fungal SDs were also predicted to be in the cytoplasm. This is in agreement with the hypothesis that the melanin precursors were synthesized in cytoplasmic organelles or in the cytoplasm (Bell and Wheeler, 1986).  3.4.2 Confirmation of O. floccosum OSD1 gene function  In order to confirm the function of OSD1 in O. floccosum, genetic disruption of this gene was attempted, but failed probably due to the very low frequency of homologous recombination that occurs in this organism. Therefore, the functional analysis of OSD1 was conducted by genetic complementation. In brown or black plant pathogenic fungi, melanin genes from the D H N pathway of one species are often able to replace their homologues in another species. For example the A. alternata ALM gene encoding a PKS can restore the melanin production and pathogenicity of the C. lagenarium mutant Pks" (Takano et al., 1997a) and the M. grisea mutant Alb" (Kawamura et al., 1997), both of which lack P K S activity. A reductase gene of A. alternata was also able to restore the melanin production and pathogenicity of the M. grisea mutant buf defective in a H N  88  reductase activity (Kawamura et al., 1997). However, this work is the first demonstration of a SD gene from one species replacing its homologue in another species.  The complementation experiment showed that OSD1 restored the mutants' melanization and pathogenicity. The OSD1 gene was determined to be integrated into the genomes of the transformants and its transcripts were detected in the melanin-restored transformants. The data also suggested that the OSD1 gene product had SD activity and that its' function was similar to that of the C. lagenarium SD. Southern blot analysis indicated that random integration of the complementation vector occurred in the genomes of the pigmentrestored transformants. Since the band intensities were different for each transformant, tandem integration may have occurred. The presence of the OSD1 gene and gene transcripts suggested that the OSD1 gene can be expressed in C. lagenarium during vegetative growth and was functional in hyphal melanization, and that its product functioned in appressorial melanization and restored the pathogenicity in C. lagenarium.  SDs of M. grisea, C. lagenarium and A. fumigatus are known to be involved in the D H N melanin pathway responsible for fungal pigmentation. A scan of N C B I nr database and est database using OSD1 did not result in finding other homologues except the three reported DHN-melanin SDs. Thus, SD gene sequences may be unique and be used as a genetic marker of the D H N melanin biosynthetic pathway. Cloning of a SD in O. floccosum suggests that the presence of a biosynthetic pathway for pigment production in 387N similar to the DHN-melanin pathway. The following evidence also supports this conclusion: 1. the recent cloning of a reductase gene which is able to complement and  89  restore the melanization of a M grisea reductase deficient mutant (Eagen, 1999). 2. the inhibition of pigmentation of 387N by tricyclazole, a DHN-melanin pathway inhibitor (Eagen, 1999). Actually, many sapstaining fungi may use the D H N pathway to synthesize melanin. Recently, a colleague has identified three major D H N melanin genes, PKS, SD and H N reductase genes in 6 other sapstaining species (Fleet, 2001).  In conclusion, a gene involved in melanin biosynthesis was isolated and characterized from the sapstaining fungus, O. floccosum. Comparative sequence analysis shows that the cloned O. floccosum gene encodes for a protein highly homologous to other fungal SD sequences. The isolated OSD1 gene restored the wild-type phenotype of the C. lagenarium mutant 9201Y, which is defective in SD activity. This indicates that the O. floccosum gene product has the same function as the SD in C. lagenarium.  90  Chapter 4  Function analysis of the second melanin reductase gene in O. floccosum  387N  4.1 Introduction  In the D H N melanin biosynthetic pathway 1,8-DHN is formed after two reduction and dehydration reactions of the starting molecule, 1,3,6,8-THN (Figure 1.4). Among the two reduction reactions, one reaction converts 1,3,6,8-THN to scytalone while the other converts 1,3,8-THN to vermelone (Figure 1.4). However, in most fungi, it is not known whether one or two reductases are necessary to catalyze these two reactions. The functional expression of the P-galactosidase-fused reductase of M. grisea shows that the fusion enzyme is able to reduce both 1,3,6,8-THN and 1,3,8-THN (Vidal-Cros et al., 1994). A few years later, a reductase purified from M. grisea was shown to be able to reduce both 1,3,6,8-THN and 1,3,8-THN (Andersson et al., 1996; Andersson et al., 1997a, 1997b). The authors stated that these results support the one-reductase hypothesis. However, this hypothesis cannot be generalized to all the fungi producing melanin through the D H N pathway. In V. dahliae, two reductase mutants have been isolated; one mutant lacks both reductase activities whereas the other lacks only 1,3,8-THN reductase activity (Bell and Wheeler, 1986).  Similarly, molecular data about the melanin pathway reductases does not support the onereductase hypothesis. Genes encoding 1,3,8-THN reductases have been cloned in M.  91  grisea, A. alternata, C. lagenarium and C. miyabeanus (Vidal-Cros et al., 1994; Shimizu et al., 1997; Kawamura et al., 1999). Disruption of the C. lagenarium THR1 gene resulted in the accumulation of a shunt product, 3,4-dihydro-4,8-dihydroxy-l  naphthalenone  (DDN), which is derived from 1,3,8-THN. This suggested that the mutant synthesized 1,3,8-THN, even though THR1 was not functional, and therefore another reductase catalyzed the reduction of 1,3,6,8-THN (see Figure 1.4). In A. fumigatus, the disruption of the reductase gene arp2 blocked the production of scytalone, indicating that this gene codes for a 1,3,6,8-THN reductase (Tsai et al., 1999). Chemical feeding and analysis of arp2 deletants have suggested that two reductase genes are present in this pathway, although the second reductase gene, converting 1,3,8-THN into vermelone, has not yet been isolated in this fungus.  Recently, a M. grisea reductase gene encoding a 1,3,6,8-THN reductase (4HNR) has been reported (Thompson et al., 2000). Mutant analyses showed that this 4HNR was only able to convert 1,3,6,8-THN to scytalone, while the 1,3,8-THN reductase (3HNR) of this fungus reduced both 1,3,8-THN to vermelone and 1,3,6,8-THN to scytalone. In O. floccosum, a reductase gene (THN1) has been isolated using a PCR-based cloning strategy. The isolated gene was found to be able to complement and restore the melanization of the M. grisea buf mutant, which lacks the gene encoding the 1,3,8-THN reductase (Eagen, 1999). During the isolation of the O. floccosum THN1, two different sequences were obtained from the amplification of the O. floccosum genomic D N A using the degenerate primers (Eagen, 1999). Both sequences showed high homology to known  92  fungal reductase genes, suggesting that two reductase genes may be present in O. floccosum.  In this chapter, the isolation and characterization of the second O. floccosum reductase gene (THN2) is presented. The role of this gene in melanin production is demonstrated by complementing the M. grisea buf mutant, a double mutant and a C. lagenarium THR1 reductase deficient mutant.  4.2 Materials and methods  4.2.1 Mutants used for complementation studies  The M grisea buf mutant IC4-1 was received from Dr. M . Farman, Department of Plant Pathology, University of Kentucky. This mutant was recovered from crosses between parental strains of Guyl 1 and 2638. The buf locus on chromosome 2 near the marker CH3-24H (Nitta et al., 1997) of strain 2638 is unstable due to the presence of many transposable elements. This locus is spontaneously deleted during mating with G u y l l , and mutations are caused by deletions of approximately 50 kb that remove all of the 1,3,8-THN reductase gene sequence. These strains were cultured on oatmeal agar at 2528°C in the dark. The M. grisea double mutant 444-5-3 lacking both 1,3,6,8-THN reductase and 1,3,8-THN reductase was provided by Dr. B. Valent from DuPont Agricultural Products, U S A . The Thr" mutant 9141 of C. lagenarium was obtained from  93  Dr. Y . Kubo at Kyoto Prefectural University, Japan. It cannot convert 1,3,8-THN to vermelone in the melanin biosynthetic pathway (Perpetua et al., 1996).  4.2.2 Plasmids  The plasmid pTHN2 was produced by ligating a 3 kb Pstl O. floccosum  genomic  fragment containing the putative reductase gene into pBSKII+. As well the 3 kb Pstl genomic D N A fragment containing the putative reductase gene was cloned into Pstl digested pCB1004 plasmid to produce the hygromycin-resistant transformation vector, pETHN. The transformation vector, pCB1.9X, which contains the full length THN1 gene was obtained from Eagen (this lab). pETHN was used to complement the M. grisea and C. lagenarium  mutants, while pCB1.9X was used to complement the M. grisea mutants.  4.2.3 Molecular techniques  D N A and R N A manipulations were the same as described in Sections 2.2.6 and 3.2.2. RT-PCR analyses, D N A sequencing and sequence data analysis using bioinformatics tools were the same as described in Sections 2.2.9, 3.2.3, 3.2.6 and 3.2.8. RT-PCR detection of the O. floccosum reductase genes' transcripts i n transformants used the primer sets R T H N 0 1 (5' G A A GTT GTC A A G C T G TTC G A 3') and R T H N 0 2 (5' CGT A C A TGT C C G TCT T G A C A 3') for THN1 and T H N R T 1 (5' T C C C A G C A A G T A C G A C G C C A T TC 3') and THNRT2 (5' C T G G C C A T C C T G C G A C G C T A G 3') for the second reductase gene.  94  4.2.4 Cloning of the second reductase gene (THN2)  in O. floccosum  To retrieve the second putative reductase gene from 387N, we aligned and compared the two insert sequences from pGT2 and pGT5 (Eagen, 1999) using the B L A S T 2 program at the National Center of Biotechnology Information (NCBI). Specific primers based on the unique region of the pGT2 insert sequence were designed and named as THN3 (5' G A C T C G T C G G C C C A G G A G GTT G T C G A 3') and THN4 (5' G A A C T G G C C G C G GGT GTT G A C G G A G A 3'). These primers and O. floccosum genomic D N A were used to perform PCR reactions (Section 2.2.7). The obtained amplicon was then used to screen the genomic library and clone the gene using procedures described previously (Section 3.2.4).  4.2.5 M. grisea and C. lagenarium protoplast formation and transformation  The protocol for Ophiostoma protoplasting and transformation (see Sections 2.2.3 and 2.2.4) was used for the transformation of M. grisea and C. lagenarium. About 40 pg of the transformation vector, pETHN, was used to transform both C. lagenarium  and M.  grisea buf mutants. The M. grisea double mutant 444-5-3 was transformed with pETHN, pCB1.9X and with both of these vectors together. For color observation, transformants of M. grisea and C. lagenarium  were transferred onto M E A and P D A , respectively.  Pathogenicity tests of C. lagenarium transformants were the same as described in Section 3.2.7.  95  4.2.6 Genomic Southern hybridization analysis  Genomic D N A from four M. grisea and three C. lagenarium  melanin-restored  transformants was digested with EcoRl and Hindlll and transferred onto Zeta-Probe GT blotting membrane (Bio-Rad). EcoRl does not cut the putative second reductase gene, while Hindlll has one cleavage site in the gene. The putative second reductase gene was used as a probe to perform the Southern blot analysis. D N A hybridization was performed under conditions of high stringency using the procedure described in Section 2.2.9. EcoRl  digested genomic D N A of O. floccosum wild type strain, M, grisea buf mutant and  C. lagenarium  mutant 9141 were included as controls. The genomic D N A from 6  transformed M. grisea double mutants was iscoRI-digested and hybridized with both O. floccosum  THN1 and THN2. EcoRl does not cut either THN1 or THN2.  4.2.7 Assay of appressorium formation  To examine conidial germination and appressorium formation, conidia were harvested from 7-day-old cultures grown on P D A medium for C. lagenarium and 5-day-old cultures grown on oatmeal agar for M. grisea. Twenty microliters of conidial suspension (10 conidia/ml for C. lagenarium and 10 conidia/ml for M. grisea) were placed onto a glass slide and incubated in a humid environment at 24°C in the dark. After 24 hr of incubation, samples were observed by microscopy (Axioplan, Carl Zeiss). Pictures were taken using a digital video camera (MDS 100, Kodak). The percentage of germinated conidia and appressoria formed were recorded for the three slides.  96  4.3 Results  4.3.1  Isolation of the putative second reductase gene in O. floccosum 3 8 7 N  We compared the insert sequences of pGT2 and pGT5 and found that -100 bp of the 400 bp sequences were identical (Figure 4.1). Therefore, specific primers based on the unique regions of the pGT2 insert sequence were designed and named as THN3 and TFIN4. PCR reactions with these primers and O. floccosum genomic D N A amplified only one band of about 170 bp (Figure 4.2). To confirm that the PCR-amplified fragment was a portion of the pGT2 insert, the P C R product was hybridized with the chemical fluorescein-labelled pGT2 insert. The P C R fragment was found to hybridize with the pGT2 insert. This suggested that the amplified PCR product was a portion of the putative second reductase gene. To isolate the full-length gene, the PCR-amplified D N A fragment was used as a probe to screen the O. floccosum genomic library. Three positive recombinant lambda phage plaques were retrieved. D N A purified from one positive recombinant lambda phage was digested using Xbal, PstI, EcoRI, EcoRN  and BamHI. Southern analysis  performed on the digested D N A using the PCR product as a probe identified a 3 kb PstI fragment containing the putative reductase gene (Figure 4.3A and 4.3B). This fragment was therefore sub-cloned into pBSKII+ (designed as pTHN2) and sequenced.  97  pGT2  pGT5  Query: 4 aaagtggcgctggtgacgggagcgggccgtggtattggtcgcggtattgccaccgagctt 63 M I  r i m M m i  m i n i !  ii i n n m i  II I I I  i n n  Sbjct: 4 aaagtggcgctggtgacgggcgcgggccgcggcattggccgcgagatggccctggagctc 63 Ouery: 64 ggccgtcgtggcgcaaatgttattgtcaacta 95  ii ii ii u m ii ii m i n i m i  Sbjct: 64 ggacgccgcggcgccaaggtcattgtcaacta 95  Figure 4.1. A nucleotide sequence comparison of the two insert sequences of the plasmids, pGT2 and pGT5 using the B L A S T 2 program. Conserved regions are indicated by boxes and the alignment is shown. Query and Subject represents the insert sequences of the pGT2 and pGT5, respectively.  1 kb 500 bp  Figure 4.2. P C R products from O. floccosum 387N genomic D N A using T H N 3 and THN4 as primers. The lane labeled M is the one kilobase ladder (BRL).  98  4 B  9  m  m  Figure 4.3. Identification of the D N A fragments containing THN2. (A) Gel patterns of the positive phage D N A digested with BamHI (lane 1), EcoRl (lane 2), EcoRV (lane 3), Pstl (lane 4), and Xbal (lane 5), and (B) identical blots hybridized with the PCRamplified fragment.  4.3.2 Sequence characterization of the second reductase gene in O. floccosum  A total of 1444 nucleotides were sequenced from the 3 kb Pstl fragment and designated as O. floccosum reductase gene, THN2 (GenBank accession number: AF317668), after the first reductase gene isolated by Eagen (1999), THN1. THN2 contained 395 bp of 5' flanking D N A , the upstream sequence from the translation initiation codon (ATG), a 855 bp open reading frame (ORF), and 194 bp of 3' downstream sequence from the stop codon (TAG) (Fig. 4.4). The G C content of the coding sequence was 63%. Several motifs  99  possibly involved in the transcription of the gene were identified in the promoter region. Putative T A T A boxes were predicted at positions -240, -254 and -341. Three putative binding sites for the transcription factor Nit2 were found at positions -106, -137, and 364. Another motif, C C A A T box was found at position -381. A potential polyadenylation signal, A A T A T A T A A A , was present 73 bp downstream from the stop codon.  THN2 consisted of an ORF without any introns and encoded a predicted protein of 284 amino acids (Figure 4.4), having a theoretical isoelectric point of 7.0 and a predicted molecular mass of 30.5 kDa. Ala, Gly and Val appeared to be the three major amino acids in the protein sequence. The frequencies of these amino acids in the THN2 deduced protein sequence were about 2-4% higher than in an average protein, while the frequencies of Glu and Leu were around 2%> lower than in an average protein (Table 4.1). The percentage of hydrophilic amino acids comprising the sequence was 44.7%. Protein solubility (S) was 1.46. The THN2 deduced protein was predicted to be located in the cytoplasm.  4.3.3 Comparison of the deduced THN2 protein with other fungal melanin reductases  To determine the association of the O. floccosum THN2 to other melanin reductases, an amino acid sequence identity matrix was constructed for the eight complete melanin reductase sequences available for the ascomycetous fungi using the clustalW alignment program and GeneDoc program (Table 4.2). The BLOSUM62 scoring matrix (Henikoff  100  and Henikoff, 1992) was used to calculate the global identity between each pair sequences.  - 3 90 tagagctcaccaatagcagcaccacaagagttatc -3 60 c g a c t t g g c a g a c g t g c a a g t a t a g t a g a c a g a a a t a c a t c t c a g -315 a t c c a t c a t g g g c t t c t c c a a c a c c g t c c g c g g c t c t c c a t g c t c -2 70 t t c c a t g a a g a g a c g g a t a t a c g g t g g c t g t a t a c c a g t a a g c c c -225 t g g c g c g g g t g t a a a t a a a a g a g g c t c t c c c c t c c a a t a t t g t t t -18 0 t c t c g a t a c t c g t a t t a t t c a c a g c c t c a c a t c t t c a t c a a c a t a -13 5 t c c t c t c a a c c t c t t c a a c a c c t c a g c a c t a t c a c c g t c a t c c t g -90 g c g a g c t t g a t a g a c t a c c t c a t a c a g g c c t t c c t c c g t c t g t a c -4 5 c a c t c g c t c t t c a c a a c a t c c c c t t c a t c a c a t a c c a c c g c c a a a 1 atggttaccacaaagtcagacaaggcccatactcccagcaagtac M V T T K S D K A H T P S K Y 4 6 gacgccattccgggcccgctcggtctgccgtcggcgtcgctggcc D A I P G P L G L P S A S L A 91 ggcaaggttgccctcgtcacgggcgcgggccgcggcattggccgc G K V A L V T G A G R G I G R 136 gagatggccctggagctcggacgccgcggcgccaaggtcattgtc E M A L E L G R R G A K V I V 181 a a c t a t g c c a a c a g c g a c t c g t c g g c c c a g g a g g t t g t c g a t g c c N Y A N S D S S A Q E V V D A 22 6 atcaaggcggccggctccgacgccgccgctattaaggccaacgtc I K A A G S D A A A I K A N V 2 71 t c c g a c g t c g a c c a g a t t g t c a c c c t c t t t g a a a a g a c c a a g c a g S D V D Q I V T L F E K T K Q 316 c a g t g g g g c a a g c t t g a c a t t g t g t g c t c c a a c t c g g g c g t c g t c Q W G K L D I V C S N S G V V 3 61 a g c t t t g g c c a t g t c a a g g a t g t c a c g c c c g a g g a g t t t g a c c g c S F G H V K D V T P E E F D R 4 06 g t c t t c t c c g t c a a c a c c c g c g g c c a g t t c t t t g t c g c g c g c g a g V F S V N T R G Q F F V A R E 451 g c c t a c a a g c a c c t c g a g a t t g g c g g c c g t c t g a t c c t c a t g g g c A Y K H L E I G G R L I L M G 4 96 t c t a t t a c c g g c c a g g c c a a g a t g g t c c c g c g g c a c g c c g t c t a c S I T G Q A K M V P R H A V Y 541 t c g g c t a g c a a g g g c g c c a t c g a g a c c t t t g t g c g c t g c a t g g c c S A S K G A I E T F V R C M A 586 g t c g a c t t t g g c g a c a a g a a g a t c a c t g t c a a c g c c g t c g c g c c t V D F G D K K I T V N A V A P 631 g g c g g t a t c a a g a c g g a c a t g t a c c a c g c t g t t t g c c g c g a g t a c G G I K T D M Y H A V C R E Y 67 6 atccccaacggcctgactctcaacgacgacgagacggatgagtac  101  I P N G L T L N D D E T D E Y 721 g c t g c t g g c t g g t c g c c c a t t c a c c g c g t c g g c c t g c c c a t t g a t A A G W S P I H R V G L P I D 766 g t g g c g c g c g t c g t t g c g t t c c t a g c g t c g c a g g a t g g c c a g t g g V A R V V A F L A S Q D G Q W 811 a t c a a c g g c a a g g t c c t t g g t g t c g a c g g c g g t g c c t g c a t g t a g I N G K V L G V D G G A C M * 856 901 94 6 991 1036  Acaatgaagggtatcacgccatggtttcgcagacaagcagtttat aaatgaatatacgtttagcaagatttgaatatataaagttacata agtgaaccataaatgtttctatacgctgctgatacaaaattctaa tatattttaacccttttaaaacttttgaacgctttttcgcccaat atacaaaccagaca  Figure 4.4. Nucleotide and deduced amino acid sequences of the O. floccosum THN2. Numbers in the left margin indicate position relative to the nucleotide of the start codon, or amino acid position. The start codon (atg) and stop codon (tag) are bolded. The putative binding site of transcription factor nit2 in the 5' untranslated leader sequence is in italic, bolded and underlined. Putative T A T A box is bolded and underlined. C C A A T box is underlined. The potential polyadenylation signal sequence is in italic and bolded.  Pairwise comparisons of the reductase sequences of O. floccosum THN2 (OfTHNB), C. lagenarium THR1 (C1THN; Perpetua et al., 1996), C. heterostrophus Brnl (ChTHN; Shimizu et a l , 1997), A. alternata BRM2 (AaTHN; Kawamura et al., 1999), and M. grisea 3HNR (MgTHNB) yielded 62-95% identity. In contrast, pairwise comparisons of each of these 5 sequences to the O. floccosum THN1 (OfTHNA), A. fumigatus arp2 (AfTHN; Tsai et al., 1999) and M. grisea 4HNR (MgTHNA) reductases yielded 42-46% identity. Therefore, these 8 reductases were divided into two groups; Group I consisted of the 3 sequences, OfTHNA, A f T H N , and M g T H N A , sharing 50-74% identity to each other, while Group II consisted of the other 5 sequences sharing 62-95% identity to each other. From this grouping it was deduced that the THN2 reductase belonged to Group II, while the THN1 reductase belonged to Group I.  102  Table 4.1. Inferred amino acid composition of O. floccosum THN2 deduced protein. Amino acid Ala (A) Arg(R) Asn (N) Asp (D) Cys(C) Gin (Q) Glu (E) Gly(G) His (H) lie (I) Leu (L) Lys(K) Met (M) Phe(F) Pro (P) Ser(S) Thr(T) Trp (W) Tyr(Y) Val (V)  No. 33 13 9 19 4 8 12 30 6 17 16 18 7 9 10 18 14 3 7 31  Real frequency (%) 11.6 4.6 3.1 6.7 1.4 2.8 3.2 10.1 2.1 6.0 5.6 6.3 2.5 3.2 3.5 6.3 4.9 1.1 2.5 10.9  Expected freq  8.8 4.2 4.6 5.9 2.1 3.7 5.9 8.3 2.1 5.4 8.0 6.3 2.0 3.7 4.5 6.6 6.0 1.4 3.7 7.1  To further examine the relationships of these reductases, a cladogram was constructed using these reductase sequences and a reductase of Streptomyces cyanogenus (GenBank accession number: AAD13552.1) as an outgroup to root the tree. S. cyanogenus reductase was assumed to have diverged early in the evolution of these reductases.  103  Table 4.2. Amino acid sequence identity matrix (%) of fungal reductases of O.  floccosum THN1 (OfTHNA) and THN2 (OfTHNB) with C. lagenarium THR1 (C1THN), M. grisea 3HNR (MgTHNB) and 4HNR (MgTHNA), A. alternata BRM2 (AaTHN), C. heterostrophus Brnl (ChTHN) and A. fumigatus arp2 (AfTHN). Sequences  OfTHNB  C1THN  MgTHNB  ChTHN  AaTHN  OfTHNA  MgTHNB  AfTHN  OfTHNB  -  78  77  66  65  44  44  44  C1THN  78  -  78  62  62  43  43  42  MgTHNB  77  78  -  69  69  43  44  44  ChTHN  66  62  69  -  95  46  45  45  AaTHN  65  62  69  95  -  46  44  44  OfTHNA  44  43  43  46  46  -  74  50  MgTHNA  44  43  44  45  44  74  51  AtTIIN  44  42  43  45  44  50  -  Notes: Group I reductases are shaded and their identity scores are boxed and shaded.  Two clusters were clearly distinguished in the phylogenetic tree (Figure 4.5). One cluster contained the three sequences, while the other contained the other 5 sequences. In the group I reductase cluster, O. floccosum O f T H N A fell into a clad with M. grisea M g T H N A , while A. fumigatus A f T H N reductase diverged from this clad. In the group II reductase cluster the C. heterostrophus ChTHN and A. alternata A a T H N reductases formed a clad that diverged from a clad consisting of the M. grisea M g T H N B , O. floccosum OfTHNB and C. lagenarium C1THN reductases.  104  ScTHN •  MgTHNB  84  OfTHNB 88 CITHN  95  ChTHN 100 AaTHN AfTHN  77  OfTHNA 100  MgTHNA  Figure 4.5. The relationships of fungal melanin reductases. The reductase sequence of (ScTHN) was used as an outgroup. The sequences were aligned using ClustalW and the cladogram was generated using Paup 4.0b6. Bootstrap values based on 1000 replicates are shown within branch nodes. Fungal reductase sequences are: O. floccosum THN1 protein (OfTHNA), M. grisea 4HNR (MgTHNA), A. fumigatus arp2 protein (AfTHN), C. heterostrophus Brnl protein (ChTHN), A. alternata BRM2 protein (AaTHN), O. floccosum THN2 protein (OfTHNB), M. grisea 3HNR (MgTHNB), and C. lagenarium THR1 protein (CITHN). Streptomyces cyanogenus  4.3.4 Complementation of the reductase defective mutants with O. floccosum  THN2  To confirm that THN2 encoded a melanin reductase, genetic complementation of reductase deficient mutants using THN2 was performed. Putative transformants were selected on culture medium containing 200 p g / m l  of hygromycin.  Transformants were selected and transferred onto hygromycin-free plates for color evaluation. Six out of ~240 M. grisea buf transformants, and eight out of ~240 C. lagenarium transformants produced black color similar to their wild type strains (Figure 4.6). Spore germination, appressorium formation and appressorium pigmentation of these transformants were observed. C. lagenarium  105  and M. grisea  which were similar to their wild-type strains, while the mutants formed slightly less pigmented appressoria (Figures 4.7 and 4.8). The pathogenicity of the C. lagenarium melanized-transformants was tested. One-month old cucumber leaves were tested with three transformants and the mutant 9141. The three melanized-transformants caused lesions on the cucumber leaves, while the melanin-deficient mutant 9141 did not cause any lesions on the leaves (Figure 4.9). These results indicated that the O. floccosum THN2 gene was acting in C. lagenarium appressorium melanization and the restoration of the pathogenicity of the C. lagenarium mutant. So far, it appeared that both O. floccosum THN1 and THN2 can function as a 1,3,8-THN reductase. We did not investigate the pathogenicity of t h e M grisea melanized-transformants due to the lack of rice seed.  1  2  3  4  5  6  Figure 4.6. The colour of the C lagenarium and M. grisea cultures. 1, 2, and 3: wild type strainl04-T, mutant 9141 and a transformant of C. lagenarium; 4, 5, and 6: wild type strain Guy 11, buf mutant and a transformant o f M grisea.  106  A  B  0« r  Figure 4.7. Appressorium pigmentation of the melanin-restored transformants of C. lagenarium. A, B and C: appressoria of the wild type, mutant 9141 and melanized transformants, respectively; bar in C = 10 um for A to C.  Figure 4.8. Appressorium pigmentation of the melanin-restored transformants of M grisea. A, B and C: appressoria of the wild type, buf mutant and melanized transformants, respectively; bar in B = 10 um; bar in C = 10 um for A and C.  107  Figure 4.9. Pathogenicity test of melanin restored transformants of C. lagenarium, complemented with O. floccosum THN2. The cucumber leaf was inoculated with the mutant 9141 (spot 1), wild type strain 104-T (spot 2), transformants (spots 3-5) of C. lagenarium. The mutant did not cause necrotic lesions while the transformants and the wild type strain caused necrotic lesions on the cucumber leaf.  1 2 3 4 5 6 7 8 9 10  Figure 4.10. Genomic Southern blot analysis of the C. lagenarium and M. grisea mutants transformed with O. floccosum THN2 gene. Lane 1: O. floccosum 387N; lane 2: C. lagenarium mutant 9141; lane 3: M. grisea buf mutant; lanes 4-6: C. lagenarium melanin-restored transformants; lanes 7-10: M. grisea melanin-restored transformants. THN2 was used as a probe for the hybridization.  108  Southern blot analysis was performed to determine whether the complementation vectors were present in the transformants' genome. Only one band was seen in O. floccosum 387N, while no band was visible in the M. grisea buf mutant or the C. lagenarium mutant (Figure 4.10). Two to ten bands corresponding to one to five copies of the O. floccosum THN2 gene were detected in both M. grisea and C. lagenarium melanized transformants (Figure 4.10). THN2 gene expression in the melanized-transformants was investigated by RT-PCR. Total RNAs were extracted from 2-day old cultures of three C. lagenarium and M. grisea melanized-transformants. Total RNAs of the O. floccosum wild type strain, M. grisea buf and C. lagenarium mutants were included as controls. R N A samples without reverse transcriptase treatment (minus-RT) were included to detect genomic D N A contamination. The primer pair (THNRT1/THNRT2) specific for the O. floccosum THN2 sequence was used in these PCR reactions. Expected fragments of 0.7 kb were produced in the six transformants and O. floccosum, but not in the C. lagenarium mutant 9141, M. grisea buf mutant and minus-RT samples (Figure 4.11).  M. grisea double mutant 444-5-3 was transformed with O. floccosum THN1, THN2 and both genes together to investigate whether O. floccosum THN1 or THN2 can function as 1,3,6,8-TFIN reductase. Three rounds of complementation were conducted and around 1,200 transformants (about 400 for each treatment) were retrieved. A l l the transformants produced color similar to the double mutant (Figure 4. 12). Southern blot analysis was performed to determine whether the complementation vectors were present in the transformants' genome. Two transformants from each treatment were chosen to conduct the experiment. The double mutant 444-5-3 was included as a control.  109  15 14 13 12 11 10  9 8  7  6 5 4  3  2 1 M  rr~ 500 bp  Figure 4.11. RT-PCR detection of the O.floccosumTHN2 gene transcript from the C. lagenarium and M. grisea mutants transformed with O.floccosumTHN2. O. floccosum (lane 1); C. lagenarium melanin-restored transformants (lanes 2, 4 and 6), their negative controls (lanes 3, 5 and 7) and mutant 9141 (lane 14); M. grisea melaninrestored transformants (lanes 8, 10 and 12), their negative controls (lanes 9, 11 and 13) and buf mutant (lane 15).  4.12. The colour of the M. grisea double mutant cultures. T l , T2, and T l - 2 : transformants of the double mutants transformed with THN1, THN2 and both THN1 and THN2, hnr", hnrTbuf, buf, and wild type: buf, hnrVbuf and buf mutants, and wild type strain of the M. grisea.  One or more bands were observed in each transformant (Figure 4.13). THN1 and THN2 expression in the transformants was investigated by RT-PCR. Total RNAs were extracted from 2-day old cultures of the six transformants. Total RNAs of the O. floccosum wild  110  type strain and the double mutant were included as controls. R N A samples without reverse transcriptase treatment (minus-RT) were included to detect the genomic D N A contamination. The primer pairs, RTHNO1 /RTHN02 for THN1 and THNRT1 /THNRT2 for THN2 were used in these PCR reactions. No signal was produced in the mutant and minus-RT samples. A n expected fragment of 0.7 kb or 0.6 kb was produced in the transformants transformed with THN2, or with THN1 from O. floccosum. Both fragments were detected in the transformants transformed with both THN1 and THN2 (Figure 4.14).  10987654321  4.13. Genomic Southern blot analysis of the M. grisea double mutant 444-5-3 transformed with O. floccosum THN1, THN2 and both. Lane 1: M. grisea double mutant 444-5-3 hybridized with THN1; lanes 2-3: transformants transformed with THN1 hybridized with THN1; lane 4-5: transformants transformed with THN2 hybridized with THN2, lanes 6-7: transformants transformed with both THN1 and THN2 and hybridized with THN1; lanes 8-9: transformants transformed with both THN1 and THN2 and hybridized with THN2; lane 10: M. grisea double mutant 444-5-3 hybridized with THN2.  Ill  M  e  d  c  b  aM  WHO.  4.14. RT-PCR detection of the 0. floccosum reductase gene transcripts from the M. grisea double mutants transformed with the O.floccosumTHN1, THN2 and both. Lanes a and c: transcripts of THN2 and THN1 from O. floccosum; b: THN2 transcripts from three THN2 transformed transformants and their minus-RT controls, d: THN1 transcripts from three THN1 transformed transformants and their minus-RT controls; e: THN1 and THN2 transcripts from two THN1 and THN2 transformed transformants and their minus-RT controls.  4.4 Discussion 4.4.1 Characterization of O.floccosumTHN2  Two different D N A sequences sharing high homology with other fungal reductase genes were amplified by P C R from O. floccosum (Eagan, 1999). This suggested that two reductase genes might be utilized in the melanin biosynthetic pathway of this fungus. Eagan (1999) isolated one reductase gene, THN1 from O. floccosum and showed that it was involved in melanin biosynthesis. In the work presented here, the isolation of the second reductase gene, THN2, is described. THN2 encodes 284 amino acids and is the longest sequence among the known fungal reductases. In contrast, THN1 reductase encoded 269 amino acids and was one of the three shortest fungal reductases.  112  THN2 did not contain any introns, while THN1 contained 1 intron. For comparison, the other melanin reductase genes in C. lagenarium, A. fumigatus, A. alternata, C. heterostrophus and M. grisea contained either two or four introns. These results are consistent with the observation that intron numbers are highly variable in filamentous fungi (Gurr et al., 1987). Three putative binding sites for the transcription factor Nit2 were found in the upstream sequence of THN2. Nit2 is involved in the regulation of the nitrogen metabolism. Similarly, several putative Nit2 protein binding sites were detected in other O. floccosum melanin genes, THN1 and OSD1 (Eagen et al., 2000; Section 3.3.1.2). This suggested that nitrogen might regulate melanin gene expression and affect O. floccosum pigmentation. However, the Nit2 protein binding sites remain to be verified. The percentage of hydrophilic amino acids comprising the reductase sequence was 44.7%, which is lower than the average percentage of hydrophilic amino acids (47.6%) of a naturally occurring soluble protein, suggesting that the encoded reductase is a hydrophobic enzyme. It has been reported that enzymes with higher hydrophobicity have higher thermal stability (Longo and Combes, 1997). This suggested that the reductase is more stable than hydrophilic enzymes at a high temperature.  4.4.2 Two groups of fungal melanin reductases  Two groups of fungal melanin reductases have been identified using an amino acid sequence identity matrix analysis. O. floccosum OfTHNB belongs to Group II reductases; these enzymes are able to convert 1,3,8-THN to vermelone in the D H N melanin  113  biosynthetic pathway. These results are in agreement with our results showing that the O. floccosum  THN2 complemented and restored the color of M. grisea and C. lagenarium  mutants that lack 1,3,8-THN reductase activity. O. floccosum O f T H N A belongs to Group I, along with M. grisea M g T H N A and A. fumigatus A f T H N which were able to convert 1,3,6,8-THN to scytalone in the melanin biosynthetic pathway.  A cladogram of these fungal reductases indicates their possible functional relationships. As expected, the fungal reductase genes fell into two distinct clusters that were well supported by the amino acid sequence identity matrix analysis. Proteins in each cluster might share a common progenitor. OfTHNA was more closely related to M. grisea M g T H N A than to A. fumigatus A f T H N , while O. floccosum OfTHNB was more closely related to the M. grisea heterostrophus  M g T H N B and C. lagenarium C1THN than to the C.  ChTHN and A. alternata AaTHN.  A n alignment of the Group I and Group II reductase sequences shows that 6 residues (marked by asterisks) are strictly conserved in all the sequences (Figure 4.15). These residues are also conserved in the short chain alcohol dehydrogenase (SADH) family (Persson et al., 1991). This suggests that these fungal reductases belong to the S A D H family. These enzymes have broad substrate specificity and some of them have high thermal stability (Secundo and Phillips, 1996). Among the 6 amino acid residues, Ser164, Tyr-178, and Lys-182 in M. grisea M g T H N B were believed to form the active site and operate as the catalytic domain for the transfer of hydrogen from the coenzyme to the  114  naphthol substrate (Persson et al., 1991; Andersson et al., 1996). Thus, it is likely that the other melanin reductases have a similar catalytic mechanism.  Nine amino acid residues from O. floccosum OfTHNB, Ser-166, He-167, Tyr-180, Met217, Tyr-218, Cys-222, Tyr-225, Trp-244 and Met-284 (marked by .; Figure 4.15) formed the enzyme binding site (Andersson et al., 1996) and are completely conserved in Group II sequences, except for the C. lagenarium CITHN, in which Met-284 was not present. Five of the nine amino acid residues (He-167, Tyr-218, Cys-222, Trp-244 and Met-284) are not present or changed to other amino acid residues in Group I sequences. In M. grisea, it was determined that the M g T H N B reductase (group II) prefers 1,3,8-THN to 1,3,6,8-THN by a factor of 4.2, and that the C-terminal residue (Met-283) of this reductase determines the substrate specificity (Thompson et al., 2000; Liao et al., 2001a).  OfTHNB CITHN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  GKVALV T T K S D K A H T P S K Y D A I P G P L G L P •AS PGVT S Q S A G — S K Y D A I P G P L G L A s AS G K V A L V GKVALV PAVTQPRGE — S K Y D A I P G P L G P Q j AS IEQ j ras SIEQ UPS SP A TVKDAARP ;KVAII' PSADITSS GPSDA^KP GKVALV TCTYLPi 5KVALV  OfTHNB CITHN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  GVQE D j j V R GN|E|SEK GNQEISEK SKPD^SVK SKPslf  ITCJ5PN|JES  115  OfTHNB C1THN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  OfTHNB ClTHN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  OfTHNB ClTHN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  OfTHNB ClTHN MgTHNB ChTHN AaTHN OfTHNA MgTHNA AfTHN  210 208 208 193 193 194 200 190  TGQAKM| TGQAKj TGQAK. TGQAK j| TGQAK AASLS AAVMT AAGL-  HAVCRE RDVCRE HAVCRE HAVCRE HAVCRE iDENSWHi IDENSWHI RENAWRj #.#  5YAAG-W| fljjFAAG-wf 3YAASA! 31YACT-H JjYACT-W |GILK-AC 1GLAN-MN  L I DV A RV'v A L I D l A RV\ C L I D l A RV\ C Q I D l A RVK c c Q VDl A T s Dl GKEK A Dl G RHV s E Dl G K\A s  R\A.  261 259 260 244 244 246 252 242  GCDKSSSLEEflETALAS-C  ACM-  284 272 283 267 267 269 274 273  Figure 4.15. Comparison of the deduced amino acid sequence of fungal reductases. Dashes indicate gaps introduced to maintain the alignment. Sequence identity of 80% and more in a column is indicated by black shading of the conserved residues and sequence similarity is indicated by grey shading. The residues conserved in short chain alcohol dehydrogenases family are indicated as *. The residues considered to be comprising the enzyme binding site are marked by . . The residues positioned near the enzyme binding site residues and showed clear difference between Group I and Group II sequences are indicated as #. Fungal reductase sequences are: O. floccosum THN1 protein (OfTHNA), M. grisea 4HNR (MgTHNA), A. fumigatus arpl protein (AfTHN), C. heterostrophus Brnl protein (ChTHN), A. alternata BRM2 protein (AaTHN), O. floccosum THN2  116  protein (OfTHNB), M. grisea 3HNR (MgTHNB), and C. lagenarium THR1 protein (C1THN).  It is highly probable that all the reductases from Group II could prefer 1,3,8-THN to 1,3,6,8-THN as a substrate. In group I reductase sequences, the five amino acid residues He-167, Tyr-218, Cys-222, Trp-244, and Met-284 are changed or not present. This might contribute to their preference for 1,3,6,8-THN.  Seven amino acid residues of O. floccosum OfTHNB, Gly-165, Thr-168, Gly-169, Ser181, Gly-185, Val-221, Arg-223 (marked by #) were completely conserved in Group II sequences, and changed to Ser, Ala, Ala, Ala, Ala, Asp and Try in Group I sequences. Interestingly, these amino acid residues are the neighbors of some of the substrate binding residues (marked b y . ; Figure 4.15). These changes may also have a contribution for the substrate preference of the enzymes. However, it is not known whether this preference is caused by the above changes, and further 3-D structure analyses of Group I reductases are needed to provide this information.  4.4.3 O.floccosumTHN2 protein function  The function of O. floccosum THN2 was confirmed by complementing C. lagenarium and M. grisea reductase deficient mutants using THN2. The accumulation of a shunt metabolite, 3,4-dihydro-4,8-dihydroxy-l  naphthalenone (DDN), derived from 1,3,8-  T H N , was observed in the medium of the C. lagenarium mutant 9141 (Perpetua et al., 1996). This mutant produces a brown phenotype on P S A , forms non-melanized  117  appressoria on glass slides, and has lost the ability to infect cucumber leaves (Perpetua et al., 1996). The mutant 9141 can be complemented by the C. lagenarium THR1 reductase gene that restores its melanization and pathogenicity (Perpetua et al., 1996). The 1,3,8T H N reductase gene sequence was deleted from the M. grisea mutant which produces a brown phenotype on M E A . The absence of this gene sequence was confirmed by hybridization studies (Eagen, 1999).  In complementation experiments, some of the transformants produced melanized mycelia and appressoria that were similar to the wild type strains. This indicated that the genomic clone of the THN2 complemented the C. lagenarium and M. grisea reductase deficient mutants and functioned i n mycelial and the appressorial melanization. This provides evidence that THN2  does i n fact encode a  homologous reductase. Furthermore, C. lagenarium melanin-restored transformants showed pathogenicity on cucumber leaves. Similarly, O. floccosum OSD1 encoded a SD that was able to restore C. lagenarium melanization and pathogenicity. It is apparent that the melanin genes from O. floccosum can replace and function as melanin genes in C. lagenarium. This phenomenon was found in other fungal D H N melanin pathways, as the reductase gene (BRM2) of A. alternata was able to restore the melanin production and pathogenicity of the M. grisea mutant buf mutant (Kawamura et al., 1997); A. alternata ALM gene encoding a P K S restored the melanin production and pathogenicity of PKS deficient mutant of C. lagenarium (mutant Pks"; Takano et al., 1997a) and M. grisea (mutant Alb"; Kawamura et al., 1997). These data suggested that the melanin genes were able to replace their counterparts in other fungal species.  118  Southern blot analysis suggested that only one copy of the THN2 gene existed in the O. floccosum  genome. In the transformants two to ten bands were observed, representing  one to five copies of integrated THN2, since the transformant genomic DNAs were digested with EcoRl and Hindlll  that cut THN2 into two fragments. RT-PCR results  suggested that THN2 was being expressed in C. lagenarium and M. grisea during vegetative growth. The analysis of THN2 function suggested that the enzyme encoded by THN2 might be involved in the conversion of 1,3,8-THN to vermelone in O. floccosum. Furthermore, the THN2 product was predicted to be located in the cytoplasm, suggesting that the conversion of 1,3,8-THN to vermelone in O. floccosum might occur in the cytoplasm.  Because THN1 complemented the buf mutant as well (Eagan, 1999), both O. floccosum THN1 and THN2 products could function as 1,3,8-THN reductases. However, both THN1 and THN2 could not complement the M. grisea double mutant lacking M g T H N A and MgTHNB, although they integrated into the transformants' genome and were being expressed in the transformants. These results suggested that neither O f T H N A nor OfTHNB can function as M g T H N A in M. grisea, although our sequence analysis suggested that OfTHNA could have a function similar to M g T H N A . However, improper posttranslational modifications of transformed OfTHNA might not allow functionality in M. grisea,  as it is known that a posttranslational modification is made in M. grisea  M g T H N A that lacked the N-terminal Met and Ala residues of the primary translation product (Thompson et al., 2000). Therefore, proper posttranslational modifications might  119  be required for O f T H N A to have proper functionality when it is expressed and translated in M. grisea. These modifications might be involved in contributing to the enzymes stability, its' correct localization within the cells, or its' enzymatic optimal activity (Shaw etal., 1990).  In addition, the localization of the reactions such as the conversion of 1,3,6,8-THN to scytalone could take place in different organelles in different fungal species. ProtComp was used to predict the probability of the subcellular localization of these reductases. The program can be used to predict the subcellular localization of fungal proteins with a predictive accuracy of 70-90%. O. floccosum OfTHNA and A. fumigatus A f T H N were predicted to be located in the mitochondria, while M. grisea M g T H N A was predicted to be located in the cytoplasm. In addition, all Group II reductases were predicted to be located in the cytoplasm. This suggests that both M. grisea, M g T H N A and M g T H N B are located in the cytoplasm, which might explain why the M. grisea mutant lacking M g T H N A produced melanin. However, it is different in A. fumigatus, the A. fumigatus mutant lacking A f T H N did not produce melanin, this might be due to A f T H N being located in the mitochondria and the other potential reductase being in the cytoplasm.  In conclusion, a second reductase gene has been isolated from O. floccosum, and thus two reductases are involved in the O. floccosum melanin synthesis. Based on an amino acid sequence identity matrix analysis, it was shown that both O. floccosum THN1 and THN2 reductases belong to two different groups of melanin reductases. Although, both reductases appeared to function as 1,3,8-THN reductase in M. grisea, it was not possible  120  to determine whether the two reductases catalyzed the same or different reduction reactions in O. floccosum.  121  Chapter 5  Cloning a partial PKS gene and analyzing transcriptional patterns of melanin genes in O. floccosum 387N  5.1 Introduction  Nutrients play an important role in the pigmentation of sapstain fungi, including O. floccosum (Eagen, 1999; Fleet, 2001). The color of fungal mycelia ranges from white, to grey, to brown and black when O. floccosum is grown on media supplemented with different carbon and nitrogen sources (Eagen, 1999). With respect to the nitrogen source, brown hyphae were observed with globulins, while white, beige or grey hyphae were observed with ammonium chloride or tyrosine. When skim milk was used as a nitrogen source, the fungus consistently produced white mycelia (Abraham, 1995). However, when the media were supplemented with asparagine the color of fungal hyphae ranged from white to black depending on the carbon source. For example, mannose addition resulted in the darkest color while the addition of starch and glucose gave light-brownish fungal mycelia. Similarly, other sapstain fungi such as O. piceae when grown on olive oil or glycerol produced brownish mycelia (Fleet, 2001). We also observed that in O. floccosum melanin was only produced in the mycelia and not in its conidia and yeast-like cells. These observations led us to question whether the expression of melanin genes in O. floccosum is affected by the nutrients or by fungal development.  122  Except for the work on melanin gene expression in C. lagenarium there is little information about fungal melanin gene expression systems (Takano et al., 1997b). In C. lagenarium,  the transcripts of the three melanin genes, PKS1, SCD1 and THR1  accumulated one or two hours after the start of conidial germination. This transcriptional pattern was observed both in differentiating and non-differentiating conidia, although no appressorium or pigment were produced from the non-differentiating conidia.  Four genes, encoding a P K S , a SD, and two reductases, are involved in the fungal melanin D H N biosynthesis pathway (Figure 1.4). So far, only three melanin genes, a SD gene (OSD1) and two reductase genes (THN1 and THN2) have been isolated in O. floccosum.  To include the fourth melanin gene in the gene expression study, we had to  isolate at least a partial PKS gene in O. floccosum.  Fungal PKSs are large multifunctional proteins that possess up to eight types of functional domains (acyl transferase, acyl carrier protein, ketosynthase, ketoreductase, enoyl reductase, dehydratase, thioesterase, and methyltransferase). The acyl transferase domain initiates the polyketide chain-building process; ketosynthase and acyl carrier protein process chain elongation; ketoreductase and dehydratase reduce and process the keto group; and thioesterase and meth-yltransferase release the full-length chain (Khosla, 1997). Fungal melanin PKS genes have been isolated in C. lagenarium, A. fumigatus and Nodulisporium lagenarium  sp  (Takano et al., 1995; Tsai et al., 1998; Fulton et al., 1999). C.  P K S uses malonyl C o A to produce 1,3,6,8-THN (Fujii et al., 1999, 2000),  123  while A. fumigatus P K S synthesizes a heptaketide from malonyl CoA, which is then converted to 1,3,6,8-THN by the gene product of aygl (Watanabe et al., 2000; Tsai et al., 2001). C. lagenarium and Nodulisporium PKSs are more similar to each other than to the A. fumigatus  melanin PKS. PKS disruptants for these three fungal species are not able to  produce pigment and are white in culture. In this chapter, we present the isolation and characterization of a partial melanin P K S gene (OPKS1), and the transcriptional regulation of the four O. floccosum melanin genes (OPKS1, OSD1, THN1 and THN2) when grown on different media.  5.2 Materials and methods  5.2.1 PCR primers and molecular techniques  PCR primers used in this study are listed in Table 5.1. D N A and R N A manipulations were the same as described in Sections 2.2.6 and 3.2.2. RT-PCR analysis, D N A sequencing and sequence data analyses using bioinformatics tools were the same as described in Sections 2.2.9, 3.2.3, 3.2.6 and 3.2.8. Protein motif analysis was carried out with the ScanProsite software at the ExPASy proteomics tool server (www.expasy.org).  5.2.2 Cloning of a partial PKS gene in O. floccosum  To isolate a portion of the P K S gene (OPKS1) in O. floccosum, two degenerate oligonucleotides, PKS3 and PKS6, were designed based on the amino acid sequences of  124  the PKSs of C. lagenarium (GenBank accession number: S60224) and A. nidulans (GenBank accession number: Q03149). The primer PKS3 is degenerate to the conserved amino acid sequence F F N M S P R E , and the PKS6 is fully degenerate to the conserved amino acid sequence M H G T G T Q . To amplify a partial PKS gene from 387N,  Table 5.1. List of the oligonucleotides synthesized for P C R reactions. Primer name  Sequence  Remark  PKS 3  5' T T C T T C A A C A T G TC(A/T)CC(C/T)CGIGA 3'  F, for OPKS1  PKS6  5' CTGGGTACC(T/G)GT(TVG)CC(A/G)TGCA 3'  R, for OPKS1  RTPKS1  5' C G T C G A C A C C T A C T A T A T C A C 3'  F, for OPKS1  RTPKS2  5' G T C A A T G T C A A G G G G A T C G A 3'  R, for OPKS1  TFTNRT1  5' T C C C A G C A A G T A C G A C G C C A T T C 3'  F, for THN2  THNRT2  5' C T G G C C A T C C T G C G A C G C T A G 3'  R, for THN2  RTFTNOl  5' G A A G T T G T C A A G C T G T T C G A 3'  F, for THN1  RTHN02  5' C G T A C A T G T C C G T C T T G A C A 3'  R, for THN1  SD23  5' G G C C T C A A T A T C A G C A G C C T C A 3'  F, for OSD1  SD26  5' T G C G C C G A T G C A A T G G C A C T 3'  R, for OSD1  NL1*  5' G C A T A T C A A T A A G C G G A G G A A A A G 3'  F, for 25S r D N A  NL2*  5' C T C T C T T T T C A A A G T G C T T T T C A T C T 3'  R, for 25S r D N A  Notes: T represents inosine. F represents a forward primer, while R represents a reverse primer. * courtesy of S. K. Kim.  125  its genomic D N A was used as a template in the PCR reactions with the primers PKS3 and PKS6 (Section 2.2.7). Purification, subcloning and sequencing of the PCR-amplified D N A fragments were performed as described in Sections 3.2.4 and 3.2.8.  5.2.3 Culture of O. floccosum  O.floccosumconidia were produced by inoculating mycelial fragments onto sterile wood blocks and incubating for 7-10 days at 20°C. Conidia were harvested by washing the wood block surface with sterile, deionized water containing 0.01% (vol/vol) Tween 20. The solution was diluted to obtain 10 conidia per ml, and 600-pl of the conidial 6  suspension was inoculated onto cellophane (BioRad) overlaid B-media agar plates (Section 2.2.5) supplemented with different carbon and nitrogen sources. The plates were incubated at 20°C. B-media with 0.1% asparagine was supplemented with 2% (w/v) glycerol (medium 1), 2% (w/v) mannose (medium 2), 2% (w/v) olive oil (medium 3). In medium 4, the B-medium was supplemented with 2% (w/v) skim milk (Defico) only. A l l the supplemented nutrients were filter-sterilized before being added to the media. Yeast cells were produced in P M medium (see Section 2.2.1) by inoculating the germinated conidia collected from media 2 after one-day incubation. A l l the experiments were repeated three times.  5.2.4 Reverse-transcription P C R  126  For R N A preparation the procedures described in Section 3.2.2 were followed. After treatment with DNAase I, the R N A samples were used as templates to perform P C R using the 26S r D N A primer set NL1-NL2. If no P C R products were amplified, the samples were considered as free of D N A contamination, and reverse-transcription reactions were preformed as described in Section 3.2.6 using 2 pg of total R N A . P C R reactions were performed using the genomic D N A free reverse-transcribed samples, and melanin gene target primer sets, RTPKS1-RTPKS2 for OPKS1, RTHNO1-RTHN02 for THN1, SD23-SD26 for OSD1, THNRT1-THNRT2 for THN2 and the 26S r D N A primer set NL1-NL2 as an internal control. As the PCR primer pair for the OSD1 spans across the two introns of the gene it was used to detect genomic D N A contamination in each sample. P C R conditions were the same as described in Section 2.2.7. Experiments were carried out to examine the levels of the melanin genes' transcripts in conidia, yeast cells and mycelia. For transcriptional analysis, samples were collected and analyzed from mycelia grown on media 1, 2, 3, and 4 for four days, and from mycelia grown for 1, 2, 3, and 4 days on media 2. In each sample, the target genes and the r D N A were amplified using 16-30 cycles in the P C R reactions. The cycle number that corresponded to the linear phase of this P C R reaction was determined and used in a subsequent P C R of all treatments. The cycle number was determined using the following procedure: reversetranscribed treatments (Day 4 samples) were diluted 1:30 with water, then 500 pi of a randomly chosen reverse-transcribed treatment was P C R amplified using the r D N A primer set by 6-30 cycles, The P C R products were gel electrophoresed by loading the same volumes of the PCR products. In order to compare the relative transcript levels of  127  the target genes the gel loading volumes of the target gene P C R reactions were standardized by loading the same relative amount of r D N A P C R product for each sample.  5.3 Results  5.3.1  Isolation of a partial P K S gene in O. floccosum  387N  With the primers PKS3 and PKS6 and O. floccosum genomic D N A , a fragment of -720 bp in length was amplified, subcloned and sequenced (Figure 5.1; GenBank accession number: AF411603). The sequence, designated OPKS1, contained 717 bp of D N A and encoded 239 amino acids. No introns were found in the sequence. A B L A S T X scan of the sequence in the N C B I nr database found 14 hits with significant E value ( l e " 72  129  - le"  ). The deduced protein sequence shared 62-91% identity and 73-94% similarity to the  PKSs of C. lagenarium, A. fumigatus, Nodulisporium sp, and A. nidulans. This strongly suggests that the OPKS1 sequence is a fragment of a P K S homolog. Furthermore, the motif analysis revealed that the OPKS1 predicted protein sequence contain a p-ketoacyl synthase motif.  Figure 5.2 shows the P-ketoacyl synthase motif of the OPKS1 predicted protein aligned with the motifs of available fungal PKSs and a PKS from Streptomyces coelicolor. Two subclasses of PKSs, WA-type and MSAS-type have been described in the fungal kingdom. The OPKS1 P-ketoacyl synthase motif is conserved throughout most of the sequence with W A type fungal PKS sequences, and the extent of conservation with the  128  1 ttcttcaacatgtcacctcgggaggctctgcagacggatcccatgcagcgcatggcaatc F  F  N  M  S  P  R  E  A  L  Q  T  D  P  M  Q  R  M  A  I  61 accacggcatttgaggctctggagatgtctggctacgtgccaaaccgtacgccctcgacg T  T  A  F  E  A  L  E  M  S  G  Y  V  P  N  R  T  P  S  T  121 cgtctcgaccgcattggcactttctacggccagacgtcggatgactggcgcgagatcaac R  L  D  R  I  G  T  F  Y  G  Q  T  S  D  D  W  R  E  I N  181 gccgcacagtccgtcgacacctactatatcacaggtggtgtgcgcgccttcggccccggc A  A  Q  S  V  D  T  Y  Y  I  T  G  G  V  R  A  F  G  P  G  241 cgcactaattatcactttggcttcagtggccctagtctcaacattgacacggcctgctca R  T  N  Y  H  F  G  F  S  G  P  S  L  N  I  D  T  A  C  S  3 01 tcgagtgctgctgccatgaacgtcgcctgcacatctctctgggcacgtgactgtgacacc S  S  A  A  A  M  N  V  A  C  T  S  L  W  A  R  D  C  D  T  3 61 gccattatcggcggtctgtcctgtatgaccaattcggacatctttgccggcctgagtcgc A  I  I  G  G  L  S  C  M  T  N  S  D  I  F  A  G  L  S  R  421 ggccagttcctatccaagacgggcccctgcgccacatttgacaacgccgctgacggctac G 4 81  Q  F  L  S  K  T  G  P  C  A  T  F  D  N  A  A  D  G  Y  tgccgtggtgatggctgtgcgtcggtcattgtcaagcgcctcgatgatgccgaggctgat C  R  G  D  G  C  A  S  V  I  V  K  R  L  D  D  A  E  A  D  541 ggtgacaatatcctggcagttatcttaggcaccgccactaaccactcggccgatgctatt G  D  N  I  L  A  V  I  L  G  T  A  T  N  H  S  A  D  A  I  601 tccatcacgcacccgcacggtcctacccagtcaatcctttcgtcagccatcttggatgat S  I T H P H G P T Q S  I  L  S  S  A  I  L  D  D  661 gccggtgtcgatccccttgacattgactatgttgagatgcacggcaccggtactcag A  G  V  D  P  L  D  I  D  Y  V  E  M  H  G  T  G  T  Q  Figure 5.1. Nucleotide and deduced amino acid sequences of O. floccosum OPKS1.  129  MSAS-type PKS motifs is much lower. This alignment shows that Cys-21, the active site, is conserved among all the fungal P-ketoacyl synthase motifs. To examine the association of the OPKS1 protein to other PKSs, a cladogram (Figure 5.3) was constructed based on the motif sequence alignment. The PKS gene from S. coelicolor was used as an outgroup. The fungal PKS proteins grouped into two distinct clusters, one cluster contained all MSAS-type PKSs including Penicillium patulum PpMSAS, Phoma sp. PspMSAS and Penicillium freii A f M S A S , and the other cluster contained the WA-type PKS sequences.  HL0CN/ jHMgCNSl 1HL|CNS| IHLSCNSS QVgCT SjjRAKE QlgCTSFWAKDj  vSpNSJjfaQKD,  sGQQAHLA JHHGRQAHLQ IHH|RQAMRA T | L R S I R D  GKSR GESK' GET G E | G  Figure 5.2. Alignment of the p-ketoacyl synthase motif of the cloned OPKS1 PCR product with other PKS P-ketoacyl synthase motifs. Sequence identity of 80% and more is indicated by black shading of the conserved residues and sequence similarity is indicated by grey shading. The active site cysteine is indicated by an asterisk. Sequence identification codes are: OpPKS, O. floccosum OPKS1 (see Figure 5.1); CIPKS: C. lagenarium PKS1 product for melanin biosynthesis (Takano et al., 1995); PspPKS: putative melanin P K S of Phoma sp. C2932 (Bingle et al., 1999); NspPKS: Nodulisporium sp. ATCC74245 pks for melanin biosynthesis (Fulton et al., 1999); ApPKS: putative W A type ketosynthase domain of Aspergillus parasiticus (Bingle et al., 1999); AfPKS: P K S fox A. fumigatus melanin biosynthesis (Tsai et al., 1998); ApPKSL: PKS for Aspergillus parasiticus aflatoxin biosynthesis (Feng and Leonard, 1995); A n W A : conidial green pigment synthase of A. nidulans (Mayorga and Timberlake, 1992); AnST: A. nidulans pksST for sterigmatocystin biosynthesis (Yu and Leonard, 1995); PpMSAS: Penicillium patulum MS AS for 6-methylsalicylic acid synthesis (Beck et al., 1990); PspMSAS: putative MSAS-type ketosynthase domain of Phoma sp. C2932 (Bingle et al., 1999); A f M S A S : Penicillium freii Pfks for 6-methylsalicylic acid synthesis (Nicolaisen et al., 1997); ScPKS: Streptomyces coelicolor PKS for antibiotic actinorhodin (Fernandes-Moreno et al., 1992).  130  ScPKS AfPKS  86  AnWA  60  ApPKS  92  ApPKSL  99  AnST CIPKS  98  OpPKS  86  NspPKS PspPKS  PpMSAS  98 97  PspMSAS AfMSAS  Figure 5.3. The relationships of the p-ketoacyl synthase motif of fungal PKSs. The PKS sequence of Streptomyces coelicolor was used as an outgroup. The cladogram was generated using Paup 4.0b6. Bootstrap values based on 1000 replicates are shown within branch nodes. Sequence identification codes are: OpPKS, O. floccosum OPKS1 (see Figure 5.1); CIPKS: C. lagenarium PKS1 product for melanin biosynthesis (Takano et al., 1995); PspPKS: putative melanin P K S of Phoma sp. C2932 (Bingle et al., 1999); NspPKS: Nodulisporium sp. ATCC74245 pks for melanin biosynthesis (Fulton et al., 1999); ApPKS: putative W A type ketosynthase domain of Aspergillus parasiticus (Bingle et al., 1999); A f P K S : P K S for A. fumigatus melanin biosynthesis (Tsai et al., 1998); ApPKSL: P K S for Aspergillus parasiticus aflatoxin biosynthesis (Feng and Leonard, 1995); A n W A : conidial green pigment synthase of A. nidulans (Mayorga and Timberlake, 1992); AnST: A. nidulans pksST for sterigmatocystin biosynthesis (Yu and Leonard, 1995); PpMSAS: Penicillium patulum M S A S for 6-methylsalicylic acid synthesis (Beck et al., 1990); PspMSAS: putative MSAS-type ketosynthase domain of Phoma sp. C2932 (Bingle et al., 1999); A f M S A S : Penicillium freii Pfks for 6methylsalicylic acid synthesis (Nicolaisen et al., 1997); ScPKS: Streptomyces coelicolor PKS for antibiotic actinorhodin (Fernandes-Moreno et al., 1992).  In the latter, three sub-clusters were formed; one sub-cluster contained three PKSs including^, parasiticus ApPKS, A. fumigatus AfPKS and A. nidulans A n W A ; the second sub-cluster contained A. nidulans AnST PKS and A. parasiticus A p P K S L ; and finally the  131  last subcluster contained O. floccosum OPKS1, Nodulisporium lagenarium  sp.  NspPKS,  C.  C1PKS and Phoma sp. PspPKS.  5.3.2 Melanin gene expression in Ofloccosum387N  The melanization of O. floccosum mycelia on different media (media 1, 2, 3, and 4) was examined. Table 5.2 showed that after one or two days of incubation the mycelia were white on all the media. However, at day 3, slight pigmentation was observed on media 1 and 2. At day 4, the mycelia were brown or light brown in color on media 1, 2 and 3, while the hyphae were still white on media 4. The fungus had no pigmentation on media 4 even after one month of incubation.  Table 5.2. Color of O.floccosummycelia on different nutrient media. Fungal color at different incubation days Culture medium Day 1  Day 2  Day 3  Day 4  Medium 1  White  White  Very light brown  Light brown  Medium 2  White  White  Light brown  Brown  Medium 3  White  White  White  Light brown  Medium 4  White  White  White  White  Note: B-media with 0.1% asparagine supplemented with 2% (w/v) glycerol (medium 1), 2% (w/v) mannose (medium 2), 2% (w/v) olive oil (medium 3). Medium 4: B-medium supplemented with 2% (w/v) skim milk.  132  To examine the presence or absence of the melanin gene transcripts in O. floccosum conidia, mycelia and yeast cells RT-PCR reactions were carried out using the primer sets for the four melanin genes and the rDNA. In these experiments, the r D N A was used as a positive control. The results showed that although transcripts of the r D N A were detected in conidia and yeast cells, no transcripts for the four melanin genes were detected in these samples. Transcripts of the four melanin genes were, however, detected in the mycelial samples (Figure 5.4).  yeast cells  cba  4321  conidia  mycelia  4 32 1 4 32 1 M  Figure 5.4. R T - P C R detection of the transcripts of the four melanin genes in conidia, yeast cells a n d mycelia of O. floccosum. Lane M : 1 kb D N A ladder, four lanes  1, 2, 3 and 4 in each sample: OPKS1, THN1, OSD1 and THN2; a-c: mycelia, conidia and yeast cells using 26S r D N A PCR primer set.  The melanin genes' transcription in mycelia grown on media 2 at 1, 2, 3, and 4 days was examined using RT-PCR (Figure 5.5). At Day 1, among the four gene transcripts, OPKS1 accumulated to the highest level, THN2 to the lowest level, and OSD1 transcripts were produced at a higher concentration than THN1 transcripts. At Day 2, all gene transcripts were more prevalent than at Day 1; OSD1 and OPKS1 transcripts were the highest and  133  THN2 transcripts were the lowest among the four genes' transcripts. At Day 3, the transcripts of the four genes were similar and higher than at Day 2. At Day 4, the transcripts of the four genes were similar to those at Day 3.  dcba  day 4  day 3  4 3 2 1  4 3 2 1  day 2  day 1  4 3 2 1 4 3  21  bp  Figure 5.5. Time course of expression of the OPKS1, THN1, OSD1 and THN2 genes during the growth of O.floccosumon media 2. Lane M : 1 kb D N A ladder, four lanes 1, 2, 3 and 4 in each sample: OPKS1, THN1, OSD1 and THN2; 26S rDNA was used as an internal control for the 4 samples, a-d: day 1-4.  The transcriptional patterns of the melanin genes in mycelia grown on media 1, 2, 3, and 4 at Day 4 were investigated using RT-PCR (Figure 5.6). The transcripts of the four genes on media 1, 2, 4 were similar and higher than the transcripts of the four genes of the fungus grown on medium 3.  134  dl  cl bl al  a  2 1 4  321  4 3 2 1  4 3 2 1  A/r  M  500 bp  Figure 5.6. The expression of the OPKS1, THN1, OSD1 and THN2 genes during the growth of O. floccosum on B-media supplemented with different combinations of carbon and nitrogen sources. Lane M : 1 kb D N A ladder, a: medium 4, b: medium 2, c:  medium 1, d: medium 3, four lanes 1, 2, 3 and 4 in each sample: OPKS1, THN1, OSD1 and THN2; 26S r D N A was used as an internal control for the 4 samples, a l : medium 4, b l : medium 2, c l : medium 1, d l : medium 3.  5.4 Discussion  5.4.1  OPKS1  is a partial DHN melanin PKS gene  The P-ketoacyl synthase motif is one of the components of a number of multifunctional enzymes including fatty acid synthetases and PKS's. Fatty acid synthetases catalyze the formation of long-chain fatty acids, while PKSs catalyze the formation of 6-  135  methysalicylic acid in P. patulum, melanin in C. lagenarium and green conidial pigment in A. nidulans (Beck et al., 1990; Mayorga and Timberlake, 1992). Among the fungal PKSs two subclasses, WA-type and MSAS-type P K S , have been proposed. WA-type PKSs initiate the synthesis of green spore pigment, melanin, and aflatoxin (Mayorga and Timberlake, 1992). The MSAS-type PKSs catalyze the biosynthesis of 6-methylsalicylic acid, the first intermediate in the pathway leading to the mycotoxin patulin in P. patulum (Beck etal., 1990).  Because we had cloned several melanin genes in the D H N pathway in O. floccosum, we assumed that a melanin PKS gene would be present in this fungus. For O. floccosum, a B L A S T X scan of the N C B I nr database using the OPKS1 sequence found a dozen hits of PKSs with significant E values. This indicated that the OPKS1 protein belonged to the PKS family instead of the fatty acid synthetase family. However, it has been reported that multiple PKS genes could be present in one fungal genome. For example, two PKS genes had been isolated in Phoma sp., one PKS is involved in the D H N melanin biosynthesis, and the other encoded a M S A S type PKS (Bingle et al., 1999). As well this situation was reported by Geisen (1996), who used a multiplex P C R method to identify sequences homologous to aflatoxin biosynthesis genes in non-aflatoxin-producing species of Aspergillus  and Penicillium.  This observation is not unexpected, as some genera of  filamentous fungi are known to produce several polyketide metabolites, requiring more than one class of polyketide backbone.  136  Although PKSs are highly divergent in sequence, the P-ketoacyl synthase motif is the most highly conserved of the PKS motifs (Bingle et al., 1999). Therefore, this motif is particularly suitable for grouping fungal PKSs. The alignment of the motifs of the OPKS1 protein and the available fungal PKSs showed high conservation with WA-type PKSs but not with MSAS-type PKSs, suggesting that OPKS1 is a WA-type PKSs gene. This motifs' sequence determines one of the enzyme functions; therefore, the cladogram (Figure 5.3) indicated the possible function relationship between the different fungal PKSs. In the diagram the WA-type PKS and MSAS-type P K S clusters were clearly identified. This was consistent with previous studies that type I PKSs are divided into two subclasses (Hopwood and Khosla, 1992; Bingle et al., 1999). In the WA-type PKS cluster, OPKS1 falls into a sub-cluster that contained three other PKSs, Nodulisporium sp NspPKS, C. lagenarium C1PKS and Phoma sp PspPKS. NspPKS, C1PKS and PspPKS are known to be involved in the D H N melanin biosynthesis in Nodulisporium sp., C. lagenarium  and Phoma sp., respectively (Fulton et al., 1999; Takano et al., 1995; Bingle  et al., 1999). Furthermore, C. lagenarium C1PKS is a pentaketide synthase (Takano et al., 1995). This strongly suggested that OPKS1 is part of a gene that encodes a pentaketide synthase involved in D H N melanin biosynthesis. A. nidulans A n W A (Mayorga and Timberlake, 1992), A. parasiticus ApPKS (Bingle et al., 1999) and the A. fumigatus AfPKS (Tsai et al., 1998) are involved in fungal conidial pigmentation and are in another sub-cluster. It was noticed that A. fumigatus AfPKS is involved in the D H N melanin biosynthetic pathway. Unlike C. lagenarium C1PKS, A. fumigatus  AfPKS is not a  pentaketide synthase, but a heptaketide synthase, although A. fumigatus still utilizes the D H N pathway to produce melanin (Tsai et al., 2001). A. nidulans A n W A is a heptaketide  137  synthase as well, although A. nidulans does not utilize the D H N pentaketide pathway (Tsai et al., 2001). Therefore, it is understandable that A. fumigatus A f P K S falls into a sub-cluster with A. nidulans A n W A instead of with C. lagenarium CIPKS! A. parasiticus A p P K S L which is involved in aflatoxin biosynthesis (Feng and Leonard, 1995) and A. nidulans  AnST which is involved in sterigmatocystin biosynthesis (Yu and Leonard,  1995) were in the same clade.  5.4.2 Melanin gene expression  Fungal development has been correlated with the production of secondary metabolites, for example in the fungal aflatoxin biosynthetic pathway researchers observed that serial transfers of macerated mycelium of Aspergillus flavus or A. parasiticus  led to the  appearance of morphological variants which also lacked the ability to produce aflatoxin (Bennett et al., 1986). Kale et al. (1994, 1996) further examined the morphological variants of A. parasiticus and observed that these mutants had an abundance of vegetative mycelia and reduced numbers of condiophores and conidia. These mutants lacked transcripts for several aflatoxin synthesis pathway genes such as nor-1, and omtA, and failed to accumulate any of the pathway intermediates. Guzman-de-Pena and RuizHerrera (1997) also associated the loss of sporulation with loss of aflatoxin production. Melanin genes are not expressed in C. lagenarium conidia but in mycelium and appressorium (Takano et al., 1997b). Similarly, O. floccosum melanin genes' transcripts were not detected in conidia and yeast cells but in mycelia. As well it was found that pigmentation occurred in mycelia but not in conidia or yeast cells of this fungus. This  138  suggests that melanin production and the melanin genes' expression in O. floccosum are probably associated with fungal development, specifically with mycelial differentiation.  In fungal secondary product biosynthesis pathways, common regulation in pathway gene expression was observed. For example, in A. nidulans, the expression of penicillin biosynthesis genes was assumed to be coordinated by some common factors, although these factors have not been yet identified (Brakhage, 1998). In fungal aflatoxin synthesis, the expression of the pathway genes was co-regulated by a regulatory gene, aflR (Brown et al., 1999). Common regulation in C. lagenarium melanin genes' expression was also suggested (Takano et al., 1997b). In the time course study, the four melanin genes of O. floccosum were expressed as the mycelium grew. In media 2 (mannose as a carbon source) the accumulation of the melanin genes' transcripts suggested that some common regulatory mechanisms coordinated the expression of the four-melanin genes in this fungus. It seems reasonable to assume that the expression of these genes is coordinated to ensure the concomitant appearance of all of the gene products in O. floccosum.  The transcripts of the four melanin genes of the fungus grown on medium 1 (glycerol) and 2 (mannose) were slightly more abundant than on medium 3 (olive oil). This was consistent with their pigmentation. O. floccosum had a darker pigment on medium 2 than on medium 3. This suggested that different carbon sources affected melanin gene expression. Mannose and glycerol led to a slightly higher expression of the genes than olive oil. However, the statistical significant of the expression differences remains to be  139  verified by some methods such as competitive RT-PCR that can monitor gene expression quantitatively.  In fungal secondary metabolite pathways, the transcription of pathway genes is not sufficient to produce the final pathway products. In A. flavus and A. parasiticus, the transcripts of the pathway regulatory gene, aflR or pathway genes were also detected without the aflatoxin biosynthesis (Feng and Leonard, 1995; L i u and Chu, 1998). In P. chrysogenum  penicillin biosynthesis, m R N A levels of all the biosynthesis genes were  highest during rapid growth when no penicillin was produced (Renno et al., 1992). It was suggested that posttranslational regulation of the pathway genes occurred, although the mechanism of this posttranslational regulation was not clear (Brakhage, 1998; Brown et al., 1999). A similar observation has been reported with C. lagenarium. Three melanin genes encoding a PKS, a SD and a reductase were expressed in colorless C. lagenarium appressoria (Takano et al., 1997b). In our study, the transcripts of all four melanin genes were observed in mycelia grown on medium 4, in which skim milk was used as the nitrogen and carbon source, which does not support melanin production. In O. floccosum, melanin biosynthesis seems not to be repressed by the skim milk through the transcriptional regulation of the melanin genes, but probably through the posttranslational regulation of the melanin genes or the inhibition of the activities of the enzymes in this pathway.  Overall, in O. floccosum, the carbon source, mannose, may support higher melanin gene expression, while skim milk repressed melanin production but not melanin gene  140  transcription. The four melanin genes' expression patterns were similar as O. floccosum grew, suggesting some common mechanisms regulate the pathway gene expression.  141  Chapter 6  Conclusions and future work  6.1. Conclusions  In order to address an important economic problem for the wood industry, wood sapstain, we proposed to explore the genetic information on the pigmentation in sapstain fungi. The discoloration of sapwood is mainly caused by sapstain fungi, which grow on wood and produce dark or brown pigment. The ultimate goal of the research program is to develop sapstain control methods by using physiological, chemical and molecular information about the growth and pigmentation of sapstain fungi.  Information was available on the physiology and biochemistry of some aspects of growth and pigmentation in the sapstain fungi. The fungal utilization of wood nutrients and the effects of some nutrients on fungal pigmentation were well described for the model organism O. floccosum 387N (Abraham et al., 1993; Abraham, 1995; Gao, 1996; Eagen, 1999). The pigment produced by the sapstain fungi has been characterized and identified as melanin. Preliminary investigations on O. floccosum melanin production included inhibitor studies and cloning of a melanin reductase gene (THN1) (Eagen, 1999). D N A sequence analyses and the genetic complementation studies indicated that O. floccosum synthesizes its melanin through a pathway similar to the DHN-melanin pathway found in many brown and black fungi. The notion of the presence of a D H N melanin biosynthetic  142  pathway in O. floccosum was strengthened by the previous observations that the color of the wild-type strain was reduced when treated with D H N pathway inhibitors, tricyclazole, carpropamid and cerulenin (Eagen, 1999; Fleet, 2001). However, the information on the pathway was incomplete, and it was necessary to generate more information on the other genes and enzymes involved in pigment production. We anticipated that information at the molecular level would help us determine whether the pigment could be controlled by different means such as designing chemicals to inhibit the pathway enzymes, or preventing wild type strain growth by inoculating albino strains, which could be produced by knocking out some pathway genes.  In this project, we isolated and characterized in O. floccosum a scytalone dehydratase gene, a second reductase gene, and a partial polyketide synthase gene. At present four genes designated OPKS1, OSD1, THN1 and THN2 have been identified and characterized. According to the deduced amino acid sequence data, the OPKS1 and OSD1 gene products are homologs of polyketide synthase and scytalone dehydratase, respectively; THN1 and THN2 gene products are homologs of H N reductase. Furthermore, the functions of OSD1, THN1 and THN2 were determined by genetic complementation of appropriate melanin deficient mutants. The OSD1 gene product was able to convert scytalone to 1,3,8-THN, and then restored the pigmentation of a fungal mutant lacking scytalone dehydratase activity; meanwhile, the THN1 and THN2 gene products were involved in another D H N melanin reaction, converting 1,3,8-THN to vermelone and restoring the pigmentation of fungal mutants lacking 1,3,8-THN reductase  143  activity. The expression of O.floccosummelanin genes, OPKS1, THN1, OSD1 and THN2 may be associated with mycelial differentiation.  Two reductase genes have been isolated in O. floccosum. In complementation studies, both the reductases of O. floccosum function to convert 1,3,8-THN to vermelone in M. grisea, but neither convert 1,3,6,8-THN to scytalone. Whether they would function in a similar way in O. floccosum remains unknown. Nevertheless, O. floccosum provides the material to study two reductases in the D H N melanin pathway.  6.2 Future directions  Targeted disruption of either and both reductase genes in O. floccosum would provide a way to elucidate the functions of both reductases in vivo. A transformation system has been set up for O. floccosum 387N, however, target gene disruption in O. floccosum failed. Therefore, further improvements of the target gene disruption in O.floccosumare necessary. Constructing a different gene disruption vector or using the A. tumefaciensfacilitated transformation could achieve the improvements. Agrobacterium-mediated transformation has been demonstrated to facilitate homologous D N A integration (Abuodeh et al., 2000); for example, Coccidioides immitis was successfully transformed by A. tumefaciens but not by other  transformation methods (Yu and Cole, 1998).  Furthermore, most A. tumefaciens-facWiXaXQd transformants contain only a single copy of integrated T-DNA (de Groot et al., 1998).  144  Direct testing of the substrate specificities of both reductases with respect to 1,3,6,8-THN and 1,3,8-THN would provide another way to understand the gene functions of O. floccosum  reductases. However, these two substrates are unstable (Bell and Wheeler,  1986) and this makes it difficult to conduct these tests. Recently, the reverse reactions of the reductases using scytalone and vermelone have been demonstrated in M. grisea (Thompson et al., 2000). In this study, the kinetic parameters of both reductases using scytalone and vermelone as substrates were measured and used to calculate the substrate preference for 1,3,6,8-THN and 1,3,8-THN. This approach could be used in O. floccosum reductases in future studies. To conduct these tests, it is necessary to express both reductase genes, purify the enzymes, synthesize scytalone and vermelone, and set up the reaction conditions and the reaction rate detection system.  The preliminary studies of melanin gene expression in O. floccosum  raise many  interesting questions: are the melanin genes co-regulated? Do some factors regulate mycelium differentiation and melanin gene expression? How might the melanin gene expression correlate with other metabolite pathways, which are involved in nutrient utilization? With current microarray technology, it should be possible to identify the genes responsible for these regulations and relations through analyzing and clustering the microarray profiles (Jagota, 2001). For example, using D N A array technology, a genomic wide study of S. cerevisiea gene expression profiles revealed many uncharacterized genes associated with regulating the metabolic shift from fermentation to respiration (Joseph et al., 1997). Given the knowledge of exogenously supplied nutrients which affect melanin biosynthesis, we could artificially perturb the biosynthesis of melanin using these  145  nutrients and use the microarray technique to look for novel genes which are altered in their regulation. We can classify and cluster the microarray gene expression profiles, which are response for melanin biosynthesis and fungal development to study the relationships between fungal development genes or their regulation genes with melanin genes.  146  References Abraham, L., 1995. 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Part 1. General characterization and the associated compounds. Holzforschung 42, 217-220.  162  Appendix  Fungal DHN melanin gene system and modeling  Biology in the 21 ' century is being transformed from a purely laboratory-based science to an information science as well. One of the crucial steps in this transformation will be training a new generation of biologists who are both computational scientists and laboratory scientists. s  - Eric  S. Lander (MIT-Whitehead  Center for  Genome  Research)  A . l Motivation  In the last ten years or so biology has become a data-rich science. One question that I am interested in involves biological modules - by that I mean groups of genes, proteins or other molecules that work together to accomplish some function at biochemical, cellular or physiological level. The fungal D H N melanin pathway is an example of the biological module. Functional genomics provide tools such as microarray analysis to allow reconstructing the biological modules (sometime called networks). This project is aimed at integrating genetic/biochemical/phenotypic information of the fungal D H N melanin pathway into the fungal genomic and microarray data (M. grisea and A. fumigatus genome sequencing are in progress, these fungi have the D H N melanin pathway) to reconstruct the whole pathway. The long-term goal of the project is to reconstruct and model the whole fungal biological networks (biological modules in the fungi) based on the fungal genomic and functional genomic data.  163  A.2 A brief description of the concepts and models  The levels of the knowledge abstraction are the following: D N A sequence to function; interaction to network to function. Graphs are used to present the networks. G = (V, E), G, V , E represent graph (network), vertices (genes, enzymes, or metabolites) and edges (the links between vertices, or the relations between genes, enzymes, or metabolites). Therefore, we could construct a genome network representing gene locations and sequence information based on genome sequencing information, a genetic network representing the gene interactions based on functional genomic data such as microarray data, and a metabolite network representing the pathway based on the known genetic and biochemical data. We can predict or reconstruct the metabolite network by aligning genome network, genetic network to metabolite network in one organism. We also can align genome network-genome network, genetic network-genetic network and metabolite network-metabolite network across species to infer knowledge. By modeling the pathway using the gene knock out data or gene expression data, we can predict the function of the network.  A.3 Results  As a preliminary effort, a software program (Fungal Melanin Gene System, Version: 1.0.0) for managing the fungal D H N melanin gene and gene knock-out information, reconstructing metabolite network and modeling the pathway has been developed. The  164  program allows searching fungal melanin gene/enzyme/gene-knock-out information, modeling and visualizing the D H N melanin pathway and the melanin gene disruption events (Figure A . l ) . For information searching, there are three choices by searching gene, searching enzyme, or searching species. In the gene and enzyme search, the user can enter the enzyme/gene name and search for either selected species or among all the species. In the species search, the user does not need to enter any keyword; he/she should select the species name and the list for enzyme or gene (Figures A.2 and A.3). For the pathway modeling and visualization/melanin-gene-disruption, the user can select which gene will be disrupted (or none is disrupted), the pathway and the color of the phenotype will be displayed (Figures A.4 and A.5). Future improvements involve database integration such as genome and microarray data, algorithm development for data analysis, network reconstruction and visualization, and reconstruction of all biological blocks in fungi.  Fungal Melanin Gene System  [  Sana S.arcfi  :  Enzyin* 8*arch j:  Spaclas Saarch  Figure A . l . The main interface of the system.  165  h  Pathway Display  r-i -rxi  i n/yrno Search View rSearch enzyme using species and enzyme name  1  Ophiostoma floccosum  Select fungal species:  T  Enter enzyme name: Search  |  Clear  J  rSearch enzymes using enzyme name-—  j  Enter enzyme name: Hunt J  Clear  I  Figure A.2. An example interface for information searching.  Et±* D i s p l a y  information  Export XML  Fasta File  Copy  Enzyme Name: Scytalone dehydratase . S O Species Name: Magnaporthe grisea Substrate: Scytalone Reaction product: 1, 3. 8-THN Protein sequence: •Scytalone dehydratase (Magnaporthe grisea] MGSQVQKSDEITFSDYLQLMTCWEWADSYDSKDWDRLRKVIAPTLRIDYRSF| L D K L W E A M P A E E F V G MVS S K Q V L G D P T L R T Q H F I G G T R W E KVS E D E VIG YH O LRVPHQRYKDTTMKEVTMKGHAHSANLHWYKKIDGVWKFAGLKPDIRWGEFu] FDRIFEDGRETFGDK  Enzyme Name* Reductase . THN1 Species Name: Magnaporthe grisea Substrate: 1. 3. 6. 8-THN Reaction product: Scytalone Protein sequence: .Reductase [Magnaporthe grisea] MPAVTQPRGESKYDAIPGPLGPQ8ASLEGKVALVTGAGRGIGREMAMELGRR G C KVIVN YAN S T E S A E EWAAIKKN G S D AAC V K A N V G W E DIVRMFE E AVKIFGK LDIVCSNSGWSFGHVKDVTPEEFORVFTINTRGQFFVAREAYKHLEIGGRLILHil G SITG O AKAVP KH AVYS G S KGAIETFAR C MAI D M AD KKITVNWAP OOIKTDMYH lAVC R E Yl P N G E N LS N E E VD E YAAS AWS P LH R V G LP IDI AR W C F LAS N D G GVWl [TGKVIGIDGGACM  Figure A.3. A searching result example.  166  Pathway Modeling and Visualisation  Fungal DHN Melanin Pathway (Modeling and Visualization) Gene disruption test, select a gene and get a color with:  Acetate  1,3,6,8-THN  Scytalone  1,3,8-THN  Vermelone  1,6-DHN  | me last compound (tuoweabeiow) -rl  Melanin  Figure A.4. A presentation of the genetic and metabolite networks of the fungal DHN melanin pathway.  BOB  ^ P a t h w a y Modeling and Visualization  Fungal DHN Melanin Pathway (Modeling and Visualization) Gene disruption test, select a gene and get a color with:  [the last compound (showed below)  Figure A.5. Pathway modeling and function inference using the gene disruption data.  167  A.4 How to run the software and the Java source code of the software  The program has been tested on Windows 98, Windows 2000 and Windows X P . To execute the program, a Sun Microsystems Java™ 2 Runtime Environment, Standard Edition (JRE), v 1.2.2 or higher versions such as 1.3.x and 1.4.x are needed. To trigger and run the program, using the "Java Model" command. Alternatively, users can double click the executable jar file (with an icon) to run the program. Beside the Java source code for the program, the following supporting files are also necessary. The gene0.txt ~ gene8.txt files for the fungal melanin gene sequence and knock out information; the enzy0.txt ~ enzy8.txt files for the fungal melanin enzyme sequence information, and the duke0.gif ~ duke3.gif files for animation.  168  II  **********************************************************************  //Version: 1.0.0 //  // @Author: Edwin (Honglong) Wang //  // Tested on Java™ 2 Platform //  // Sun Microsystems, Inc., // 901 San Antonio Road, // Palo Alto, CA 94303 USA. //  // Copyright (c) 2000 // E. Wang // University of British Columbia, // Vancouver, Canada //  // All Rights Reserved //  // License: non-commercial II  **********************************************************************  // Sequence.java /** this class defines the information of a sequence, * the superclass of the classes: Gene and Enzyme */ public class Sequence { /** the name of the sequence */ private String seqName; /** the sequence ID number */ private String seqID; /** the sequence information */ private String seqlnfo; /** species name */ private String speciesName; /** a constructor */ public Sequence() { seqName = "unknown"; seqID = "unknown"; speciesName = "unknown";, seqlnfo = "unknown"; }  /** a convenience constructor */ public Sequence(String n, String i, String sn) {  seqName = n; seqID = i; speciesName = sn; seqlnfo = "unknown";  169  } /** the method is to set the name of the sequence */ public void setName(String name) { }  seqName = name;  /** the method is to set the ID number of the sequence */ public void setID(String id) {  }  seqID = id;  /** the method is to read the sequence information */ public void setSeq(String info)  { seqlnfo = info; }  /** the method is to set the species name */ public void setSpeciesName(String species) {  }  speciesName = species;  /** the method is to obtain the name of the sequence */ public String getName() { }  return seqName;  /** the method is to obtain the ID number of the sequence */ public String getID() { }  return seqID;  /** the method is to obtain the information of the sequence */ public String getSeq() { }  return seqlnfo;  /** the method is to obtain the species name */ public String getSpeciesName() {  return speciesName; }  // Gene Java  170  /** this class defines gene information, subclass of the class Sequence */ public class Gene extends Sequence  {  /** gene description */ private static String description; /** a constructor */ public Gene() { super(); description = "A structure gene"; }  /** a convenience constructor */ public Gene(String n, String i, String sn) { super(n, i, sn); description = "A structure gene"; } } II ******************************************  // Genelist.java import java.util.*; /** the class stores genes */ public class Genelist { /** the Genelist varaible,type: ArrayList * Gene objects will be stored*/ protected ArrayList geneCollection; /** the constructor */ public Genelist () { geneCollection = new ArrayList(); }  /** This method is to add Gene objects to the gene collection */ public void addGene(Gene c) {  if(c == null) c = new Gene(); else {if(geneCollection.contains(c) == false) geneCollection.add(c); }  }  /** This method is to remove Gene objectsfromthe collection */ public ArrayList removeGene(Gene c) {  geneCollection.remove(geneCollection.indexOf(c)); return geneCollection;  }  /** The method is to search the genefromthe list * the search attributes is the species name */ 171  public ArrayList speciesSearch(String species) { ArrayList c = new ArrayList(); int i = 0; Gene tempGene = null; for(i = 0; i < geneCollection.size(); i++){ tempGene = (Gene) geneCollection.get(i); if (species.equalsIgnoreCase(tempGene.getSpeciesName())) c.add(tempGene); }  }  return c;  /** The method is to search the genefromthe list * the search attributes is the species name and the gene name */ public Gene geneSearch(String species, String name) {  int i = 0; Gene tempGene = null; for(i = 0; i < geneCollection.size(); i++){ tempGene = (Gene) geneCollection.get(i); if (species.equalsIgnoreCase(tempGene.getSpeciesName()) && name.equalsIgnoreCase(tempGene.getName())) return (Gene) tempGene; }  return null; }  /** The method is to search the genefromthe list * the search attributes is the gene name */ public Gene nameSearch(String name) {  int i = 0; Gene tempGene = null; for(i = 0; i < geneCollection.size(); i++){ tempGene = (Gene) geneCollection.get(i); if (name.equalsIgnoreCase(tempGene.getName())) return (Gene) tempGene; }  return null; } II  }  **************************************  // GeneView.java import javax.swing.*; //import javax.swing.event.*; import java.awt. *; import java.awt.event.*; import javax.swing.border. *; /**  172  * The class is responsible for providing an interface for gene search. */ public class Gene View extends JFrame { /** declare the references. */ JComboBox cbox = null; Container c = null; JButtonbl,b2, b3, b4; JTextFieldtl, t2; /** define an array of species name to be shown in the * combo box pull-down list, by default. */ String[] defaultSpecies = { "Ophiostoma floccosum", "Magnaporthe grisea", "Colletotrichum lagenarium"}; String defaultEdit = "Ophiostoma floccosum"; /** model variable */ protected Model mymodel; /** constructor */ public Gene View (Model m) {  super(); setTitle("Gene Search View"); mymodel = m; //configure theframeusing its content pane c = this.getContentPane(); c.setBackground(Color.lightGray); c.setLayout(new GridLayout(2,l)); initO;  }  sizeandplaceWindow(); addWindowListener(new WindowCloser()); setDefaultCloseOperation(WindowConstants.DISPOSE_ON_CLOSE);  /** construct the view */ public void init() { // prepare the container with layout JPanel pi = new JPanel(); p 1 .setLayout(new BorderLayout()); // set thetitleborder around the panel. TitledBorder borderl = new TitledBorder( new LineBorder(Color.blue), "Search gene using species and gene product names"); border 1. setTitleColor(Color.blue); pi .setBorder(borderl); JPanel p2 = new JPanel(); p2. setLayout(ne w BorderLayout()); // set the title border around the panel. TitledBorder border2 = new TitledBorder( new LineBorder(Color.blue),  173  "Search genes using gene product names"); border2. setTitleColor(Color.blue); p2.setBorder(border2); JPanel p3 = new JPanel(); p3 .setLayout(new GridLayout( 1,2)); JPanel p4 = new JPanel(); p4.setLayout(new GridLayout( 1,3)); JPanel p5 = new JPanel(); p5. setLayout(new GridLayout( 1,2)); JPanel p6 = new JPanel(); JPanel p7 = new JPanel(); JPanel p8 = new JPanel(); // create the combo box with the list of default sites, cbox = new JComboBox(defaultSpecies); cbox. setOpaque(true); // make the combo box ineditable. cbox.setEditable(false); // configure the combo box editor. cbox.configureEditor(cbox.getEditor(), defaultEdit); // rows to be visible without scrollbars, cbox. setMaximumRo wCount(3); // set the combo box editor colors and font. ComboBoxEditor cboxEditor = cbox.getEditor(); Component editorComp = cboxEditor.getEditorComponent(); editorComp.setBackground(Color.white); editorComp.setForeground(Color.blue); Font fl = new FontfDialog", Font.PLAIN, 14); editorComp.setFont(fl); // font for the combo box popup list. cbox.setFont(fl); // label to indicate what to do. JLabel labell = new JLabel("Select fungal species:", JLabel.CENTER); labell.setFont(newFont("SansSerif', Font.BOLD, 16)); JLabel label2 = new JLabel("Enter gene product name: JLabel.CENTER); label2.setFont(newFont("SansSerif, Font.BOLD, 16)); JLabel label3 = new JLabel("Enter gene product name: JLabel.CENTER); label3.setFont(newFont("SansSerif', Font.BOLD, 16)); JLabel labeW = new JLabelf JLabel.CENTER); // button for search bl = new JButton("Find"); 174  bl.setFont(newFont("SansSerif, Font.BOLD, 16)); b 1 .addActionListener(mymodel); b2 = new JButton("Go"); b2.setFont(new Font("SansSerifFont.BOLD, 16)); b2 .addActionListener(mymodel); b3 = new JButton("Clear"); b3.setFont(newFont("SansSerif, Font.BOLD, 16)); b3.addActionListener(new Listener()); b4 = new JButton("Clear"); b4.setFont(new Font("SansSerif', Font.BOLD, 16)); b4.addActionListener(new Listener()); // textfiels tl = new JTextField(2); tl.setFont(fl); t2 = new JTextField(2); t2.setFont(fl); //construct thefirstsearch block p3.add(labell); p3.add(cbox); p4.add(label3); p4.add(tl); p7.add(bl); p7.add(b3); pl.add(p7, BorderLayout.SOUTH); pl.add(p4, BorderLayout.CENTER); pl.add(p3, BorderLayout.NORTH); //construct the 2nd search block p5.add(label2); p5.add(t2); p6.add(b2); p6.add(b4); 8.add(label4); P  p2.add(p6, BorderLayout.SOUTH); p2.add(p5, BorderLayout.CENTER); p2.add(p8, BorderLayout.NORTH); //add the blocks to the frame c.add(pl); c.add(p2); /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter { public void windowClosing(WindowEvent e){ dispose(); } }  /** this class is to implement the ActionListener interface */ private class Listener implements ActionListener { /** the method is listening */  175  public void actionPerformed(ActionEvent e){ Object source = e.getSource(); if (source = b3) tl.setText(""); else if (source == b4) t2.setText(""); }  }  /** this private method sizes and places theframein the middle of the screen */ private void sizeandplaceWindow() {  this.setSize(480, 290); this.setResizable(false); DimensionframeD= new Dimension(480, 290); Dimension screenD = new Dimension(); screenD = Toolkit.getDefaultToolkit().getScreenSize(); if(frameD.width>= screenD. width) this.setLocation(l, 1); this.setLocation(((screenD.width - frameD.width)/2), ((screenD.height - frameD.height)/2)); this.setSize(frameD. width,frameD.height);  }  //testing public static void main(String arg[]) { Model a = new Model(); Gene View gv = new GeneView(a); } } II  ***********************************  //Structure.java public class Structure { public Structure(){}  } II  **********************************************************************  // Enzyme .Java /** this class defines protein information, subclass of the class Sequence */ public class Enzyme extends Sequence {  /** its gene */ private Gene gene; /** the substrate name */ private String subName; 176  /** the raction product */ private String productName; /** a constructor */ public Enzyme() { super(); subName = "unknown"; productName = "unknown";  } /** a convenience constructor */ public Enzyme(String a, String b, String c, Gene g, String sn, String pn ) {  super(a, b, c); gene = g; subName = sn; productName = pn;  }  /** the method is to set the gene */ public void setGene(Gene ge) { gene = ge; }  /** the method is to set the substrate name */ public void setSubName(String substrate) {  subName = substrate; }  /** the method is to set the product name */ public void setProductName(String product) { productName = product; }  /** the method is to obtain the gene */ public Gene getGene() { }  return gene;  /** the method is to obtain the substrate name */ public String getSubName() {  return subName; }  /** the method is to obtain the product name */ public String getProductName() { }  return productName;  177  II *******************************  // Enzymelist.java import java.util.*; /** the class stores enzymes */ public class Enzymelist {  /** the Enzymelist varaible.type: ArrayList * Enzyme objects will be stored*/ protected ArrayList enzyCollection; /** the constructor */ public Enzymelist () { enzyCollection = new ArrayList(); }  /** This method is to add Enzyme objects to the enzyme collection */ public void addEnzyme(Enzyme c) {  if(c = null) c = new Enzyme(); else {if(enzyCollection.contains(c) = false) enzyCollection.add(c); }  }  /** This method is to remove Enzyme objects from the collection */ public ArrayList removeEnzyme(Enzyme c) {  enzyCollection.remove(enzyCollection.indexOf(c)); return enzyCollection;  } /** The method is to search the enzyme from the list * the search attributes is the species name */ public ArrayList speciesSearch(String species)  {  ArrayList c = new ArrayList(); Enzyme tempEnzyme = null; for(int i = 0; i < enzyCollection.size(); i++){ tempEnzyme = (Enzyme) enzyCollection.get(i); if (species.equalsIgnoreCase(tempEnzyme.getSpeciesName())) c. add( tempEnzyme); }  }  return c;  /** The method is to search the enzymefromthe list * the search attributes is the species name and the enzyme name */ public Enzyme enzySearch(String species, String name) {  Enzyme tempEnzyme = null; 178  for(int i = 0; i < enzyCollection.size(); i++){ tempEnzyme = (Enzyme) enzyCollection.get(i) if (species.equalsIgnoreCase(tempEnzyme.getSpeciesName()) && name.equalsIgnoreCase(tempEnzyme.getName())) return (Enzyme) tempEnzyme; }  return null;  /** The method is to search the enzymefromthe list * the search attributes is the enzyme's name */ public Enzyme nameSearch(String name) {  Enzyme tempEnzyme = null; for(int i = 0; i < enzyCollection.size(); i++){ tempEnzyme = (Enzyme) enzyCollection.get(i); if (name.equalsIgnoreCase(tempEnzyme.getName())) return (Enzyme) tempEnzyme; } return null; }  /** The method is to search the enzymefromthe list * the search attributes is the enzyme's name */ public Enzyme nameSearchl (String name)  {  Enzyme tempEnzyme = null; //for(int i = 0; i < enzyCollection.size(); i++) { //tempEnzyme = (Enzyme) enzyCollection.get(i); // if (name.equalsIgnoreCase(tempEnzyme.getName())) { Listlterator counter = enzyCollection.listIterator(); while(counter.hasNext()) { tempEnzyme = (Enzyme) counter.next(); if (name.equalsIgnoreCase(tempEnzyme.getName())) { //(Enzyme) tempEnzyme = counter.next(); return tempEnzyme;} }  return tempEnzyme; }  public void print() { Enzyme tempEnzyme = null; for (int k = 0; k < enzyCollection.size(); k++){ tempEnzyme = (Enzyme) enzyCollection.get(k); System.out.println(tempEnzyme.getName()); System.out.println(tempEnzyme.getSeq()); } } } II  **********************************************  // Enzyme View.java import javax.swing.*; 179  import j ava. awt. *; import java.awt. event.*; import javax.swing.border.*; import javax.swing.event.*;  * The calass is responsible for providing an interface for enzyme search. */ public class Enzyme View extends JFrame { /** declare the references. */ JComboBox cbox = null; Container c = null; JButtonbl,b2, b3, b4; JTextFieldtl,t2; /** define an array of species name to be shown in the * combo box pull-down list, by default. */ String[] defaultSpecies = {"Ophiostoma floccosum", "Magnaporthe grisea", "Colletotrichum lagenarium"}; String defaultEdit = "Ophiostoma floccosum"; /** model variable */ protected Model mymodel; /** constructor */ public EnzymeView (Model m) {  super(); setTitle("Enzyme Search View"); mymodel = m; //configure theframeusing its content pane c = this.getContentPane(); c.setBackground(Color.lightGray); c.setLayout(new GridLayout(2,1)); initO;  sizeandplaceWindow(); addWindowListener(new WindowCloser()); setDefaultCloseOperation(WindowConstants.DISPOSE_ON_CLOSE); }  /** construct the view */ public void init() { // prepare the container with layout JPanel pi = new JPanel(); pi .setLayout(new BorderLayout()); // set the title border around the panel. TitledBorder borderl = new TitledBorder( new LineBorder(Color.blue), "Search enzyme using species and enzyme name"); borderl.setTitleColor(Color.blue); pi .setBorder(borderl); JPanel p2 = new JPanel(); p2.setLayout(new BorderLayout()); // set the title border around the panel. 180  TitledBorder border2 = new TitledBorder( new LineBorder(Color.blue), "Search enzymes using enzyme name"); border2 .setTitleColor(Color.blue); p2.setBorder(border2); JPanel p3 = new JPanel(); p3 .setLayout(new GridLayout( 1,2)); JPanel p4 = new JPanel(); p4. setLayout(ne w GridLayout( 1,3)); JPanel p5 = new JPanel(); p5. setLayout(new GridLayout( 1,2)); JPanel p6 = new JPanel(); JPanel p7 = new JPanel(); JPanel p8 = new JPanel(); // create the combo box with the list of default species, cbox = new JComboBox(); for(inti = 0;i<3;i++) cbox. addItem(defaultSpecies [i]); cbox.setOpaque(true); // make the combo box ineditable. cbox. setEditable(false); Font fl = new Font("Dialog", Font.PLAIN, 14); // font for the combo box popup list. cbox.setFont(fl); // label to indicate what to do. JLabel labell = new JLabel("Select fungal species:", JLabel.CENTER); labell.setFont(newFont("SansSerif', Font.BOLD, 16)); JLabel label2 = new JLabel("Enter enzyme name:", JLabel.CENTER); label2.setFont(newFont("SansSerif', Font.BOLD, 16)); JLabel label3 = new JLabel("Enter enzyme name:", JLabel.CENTER); label3.setFont(newFont("SansSerif', Font.BOLD, 16)); JLabel labeW = new JLabel(" JLabel.CENTER); // button for search bl = new JButton("Search"); bl.setFont(newFont("SansSerif, Font.BOLD, 16)); bl .addActionListener(mymodel); b2 = new JButton("Hunt"); b2.setFont(new Font("SansSerif, Font.BOLD, 16)); b2.addActionListener(mymodel); b3 = new JButton("Clear"); b3.setFont(newFont("SansSerif, Font.BOLD, 16)); b3.addActionListener(new Listener()); b4 = new JButton("Clear"); b4.setFont(new Font("SansSerif, Font.BOLD, 16)); b4. addActionListener(ne w Listener()); // textfiels tl = new JTextField(2); tl.setFont(fl); 181  t2 = new JTextField(2); t2.setFont(fl); //construct the first search block p3.add(labell); p3.add(cbox); p4.add(label3); p4.add(tl); p7.add(bl); p7.add(b3); pl.add(p7, BorderLayout.SOUTH); pl.add(p4, BorderLayout.CENTER); pl.add(p3, BorderLayout.NORTH); //construct the 2nd search block p5.add(label2); p5.add(t2); p6.add(b2); p6.add(b4); p8.add(label4); p2.add(p6, BorderLayout.SOUTH); p2.add(p5, BorderLayout.CENTER); p2.add(p8, BorderLayout.NORTH); //add the blocks to the frame c.add(pl); c.add(p2); /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter { public void windowClosing(WindowEvent e){ dispose(); } }  /** this class is to implement the ActionListener interface */ private class Listener implements ActionListener { /** the method is listening */ public void actionPerformed(ActionEvent e) { Object source = e.getSource(); if (source = b3) tl.setText(""); else if (source == b4) t2.setText(""); }  }  /** this private method sizes and places theframein the middle of the screen */ private void sizeandplaceWindow() I  this.setSize(480, 290); 182  this .setResizable(false); DimensionframeD= new Dimension(480, 290); Dimension screenD = new Dimension(); screenD = Toolkit.getDefaultToolkit().getScreenSize(); if(frameD.width >= screenD.width) this.setLocation(l, 1); this.setLocation(((screenD.width - frameD.width)/2), ((screenD.height - frameD.height)/2)); this.setSize(frameD.width, frameD.height); }  //testing public static void main(String arg[]) { Model a = new Model(); EnzymeView gv = new EnzymeView(a); } II  }  **********************************************  // Compound.java import java. awt. *; /** this class defines pathway compound information */ public class Compound {  /** the previous compound name */ private String preName; /** the compound name */ private String compName; /** the raction product */ private String productName; /** the structure of the compound */ private Structure compStruct; /** the compound position- integer */ private int position; /** the genes associated*/ private Gene gene; /** the color of the phenotype when the compound is blocked*/ private Color color; /** a constructor */ public Compound() { preName = "unknown"; compName = "unknown"; productName = "unknown"; position = 0; gene = new Gene(); color = new Color( 1,1,1);  } /** a convenience constructor */ public Compound (String a, String sn, String pn, int n, Color c ) {  183  preName = a; compName = sn; productName = pn; position = n; color = c; }  /** the method is to set the previous compound */ public void setPreName(String ge) {  }  preName = ge;  /** the method is to set the compound name */ public void setCompName(String comp) { compName = comp; }  /** the method is to set the product name */ public void setProductName(String product) { productName = product;  } /** the method is to set the gene obj */ public void setGene(Gene g) { }  gene = g;  /** the method is to set the compound position with integer public void setPosition(int p) {  position = p; }  /** the method is to set the color */ public void setColor(Color c) { color = c;  } /** the method is to obtain the previous compound */ public String getPreName() {  return preName; }  /** the method is to obtain the compound name */ public String getCompName() {  return compName; }  /** the method is to obtain the product name */ 184  public String getProductName() { return productName; }  /** the method is to get the gene obj */ public Gene getGene() { }  return gene;  /** the method is to get the compound position with integer */ public int getPosition() {  return position; }  /** the method is to get the color */ public Color getColor() {  }  return color;  } jl  *****************************************  // CompoundSet.java import j ava.util. *; /** the class stores Compounds */ public class CompoundSet {  /** the CompoundSet varaible,type: ArrayList * Compound objects will be stored*/ protected ArrayList compCollection; /** the constructor */ public CompoundSet () { compCollection = new ArrayList(); }  /** This method is to add Compound objects to the Compound collection */ public void addCompound(Compound c)  {  }  if(c = null) c = new Compound(); else compCollection.add(c);  /** This method is to remove Compound objects from the collection */ public ArrayList removeCompound(Compound c) {  compCollection.remove(compCollection.indexOf(c)); 185  return compCollection; } /** The method is to search the Compoundfromthe list * the search attributes is the gene's name */ public Compound geneNameSearch(String name) { Compound tempCompound = null; Listlterator counter = compCollection.listIterator(); while(counter.hasNext()) { tempCompound = (Compound) counter.next(); if (name.equalsIgnoreCase(tempCompound.getGene().getName())) return tempCompound;  } return tempCompound; } /** get the elementsfronthe first elem to the c elem*/ public ArrayList getFirstPart(Compound c) { int index = getlndex (c); Compound tempCompound = null; for(int i = index + 1; i < compCollection.size(); i++){ tempCompound = (Compound) compCollection.get(i); removeCompound(tempCompound);  }  return compCollection; } public int getSize(){ return compCollection.size();  } public Compound getElement(int i){ Compound tempCompound = null; if (i <= compCollection.sizeO) tempCompound = (Compound) compCollection.get(i); return tempCompound;  } public int getlndex (Compound c) { return (int) compCollection. indexOf(c); }  } U  ****************************************  // Pathway .Java import j avax. swing .event. *; import j avax. swing. *; import java.util.*; import java.awt.*; import java.lang.*; import java.io.*;  186  import java.awt.event. *; import javax.swing.tree.*; import Java.util.*; /** this class for building the DHN pathway */ public class Pathway { /** Compounds variable */ protected Compound cp; //views will be triggered by this protected PathwayView pathwayview; String geneNames [] = {"Polyketide synthase gene (PKS)", "Reductase I gene (THN1)", "Scytalone dehydratase gene (SD)", "Reductase II gene (THN2)", "Scytalone dehydratase gene (SD)", "Oxidases"}; //compund names in the pathway String compNames[] = {"Acetate", "1,3,6,8-THN", "Scytalone", "1,3,8-THN", "Vermelone", "1,8-DHN", "Melanin"}; Gene genes [] = new Gene [6]; Compound compounds[] = new Compound [compNames.length]; Color colors [] = {Color.white, Color.pink, Color.pink, new Color(208,160,132),/*brown*/ Color.white, new Color(208,160,132),Color.black}; CompoundSet comCollect; public Pathway(int z){ comCollect = new CompoundSet(); //TreeSet is sorted/unrepeated pathwayBuilder(z); }  /** build the compounds in the pathway */ public CompoundSet pathwayBuilder(int len){ //creat genes and assign gene names for them for (int i = 0; i < len-1; i++){ genes[i] = new Gene(); genes[i]. setName(geneNames [i]); }  //creat compounds and assign gene names and positions for them for (intj=0;j<len;j++){ compounds[j] = new Compound(); compounds [j ]. setPosition(j * 10); compounds [j ]. setCompName(compNames[j ]); compounds [j ]. setColor(colors [j ]); if (j == len -1) break;//no gene is associated with the last compound compounds[j].setGene(genes[j]); }  for (int k = 0; k < len; k++){ comCollect.addCompound(compounds[k]); }  return comCollect; }  187  public int getPathwaySize(){ return comCollect.getSize(); }  public Compound getPathwayElement(int i){ Compound tempCompound = null; if (i <= getPathwaySize()) tempCompound = (Compound) comCollect.getElement(i); return tempCompound; }  public static void main (String [] args) { new Pathway(7); } } II  *************************************************  // DrawPathway .Java import java.awt.*; import java.awt.event.*; import Java.awt.geom.*; import javax.swing.*; import java.awt.event.ItemListener; import java.awt.event.ItemEvent; import java.awt.event.ActionListener; import java.awt.event.ActionEvent; import java.util.*; public class DrawPathway extends JPanel { final static int maxCharHeight =15; final static int minFontSize =11; final static Color bg = Color.white; final static Color fg = Color.black; final static Color red = Color.red; final static Color white = Color.white; final static BasicStroke stroke = new BasicStroke(2.0f); final static BasicStroke wideStroke = new BasicStroke(8.0f); final static float dashl[] = {lO.Of}; final static BasicStroke dashed = new BasicStroke(1.0f, BasicStroke.CAPBUTT, BasicStroke.JOIN MITER, lO.Of, dashl, O.Of); Dimension totalSize; FontMetrics fontMetrics; CompoundSet c; Pathway p; HashMap geneHash; public DrawPathway(Pathway pi) { //Initialize drawing colors setBackground(bg); 188  setForeground(fg); geneHash = new HashMap(); p=pl; // Compound pathwayCompounds [] = new Compoundf] { }; }  //gene names in abbrev public HashMap setGeneName(){ geneHash.put ("Polyketide synthase gene (PKS)", "PKS"); geneHash.put("Reductase I gene (TFTNl)", "THN1"); geneHash.put("Scytalone dehydratase gene (SD)", "SD"); geneHash.put("Reductase II gene (THN2)", "THN2"); geneHash.put("Oxidases", "OXD"); return geneHash; }  FontMetrics pickFont(Graphics2D g2, String longString, intxSpace) { boolean fontFits = false; Font font = g2.getFont(); FontMetrics fontMetrics = g2.getFontMetrics(); int size = font.getSize(); String name = font.getName(); int style = font.getStyle(); while ( ! fontFits ) { if ( (fontMetrics.getHeight() <= maxCharHeight) && (fontMetrics.stringWidth(longString) <= xSpace)) { fontFits = true; }  else { if ( size <= minFontSize) { fontFits = true; }  else { g2.setFont(font = new Font(name, style, -size)); fontMetrics = g2.getFontMetrics(); } }  }  return fontMetrics; }  public void paint(Graphics g) { Graphics2D g2 = (Graphics2D) g; //compound number in the pathway int pathwaySize = p.getPathwaySize(); HashMap hm; 189  hm = this.setGeneName(); g2.setRenderingHint(RenderingHints.KEY_ANTIALIASING, RenderingHints.VALUE_ANTIALIAS_ON); Dimension d = getSize(); int gridWidth = d.width / 8; int gridHeight = d.height / 8; fontMetrics = pickFont(g2, "Filled and Stroked GeneralPath", gridWidth); Color fg3D = Color.lightGray; g2.setPaint(fg3D); g2.draw3DRect(0, 0, d.width - 1, d.height - 1, true); g2.draw3DRect(3, 3, d.width - 7, d.height - 7, false); g2.setPaint(fg); intx= 10; inty = 10; // y += gridHeight; int rectWidth = gridWidth - 2*x; int stringY = gridHeight - 3 - fontMetrics.getDescent(); int rectHeight = stringY - fontMetrics.getMaxAscent() - y - 1; //stringY += gridHeight; //draw the pathway Shape compoundShpesf] = new Shape[pathwaySize]; Shape lines[] = new Shape [pathwaysize]; g2. setStroke(stroke); Compound myCompounds[] = new Compound [pathwaysize]; String compName; String geneName; Color color; draw: for(int i = 0; i < pathwaySize; i++){ compoundShpes[i] = new Ellipse2D.Double(x, y+150, rectWidth/2, rectHeight); myCompounds[i] = (Compound) p.getPathwayElement(i); compName = myCompounds[i].getCompName(); String key = null; key = myCompounds[i].getGene().getName(); geneName = (String) hm.get (key); color = myCompounds[i].getColor(); g2.setPaint(color); g2.fill(compoundShpes[i]); g2.setPaint(fg); g2.drawString(compName, x, stringY+l52); x += gridWidth/2 intyl; yl = y + 150 + rectHeight/2; // draw Line2D.Double lines[i] = new Line2D.Double(x, yl, x + rectWidth/2, yl); //the last line can not be appeared if (i == (pathwaySize-1)) break draw; g2.draw(lines[i]); g2.drawString(geneName, x, stringY+100); x += gridWidth/2; 190  } } } II  **********************************  //PathwayView.java import j ava. awt. *; import java.awt.event.*; import java.awt.geom.*; import javax.swing.*; import java.awt.event.ItemListener; import java.awt.event.ItemEvent; import java.awt.event.ActionListener; import Java.awt.event.ActionEvent; /*  * This class for displaying the DHN pathway, it * uses the Java 2D APIs to define and render the graphics and text. */ public class PathwayView extends JFrame implements ActionListener { String geneNames [] = new String [] {"the last compound (showed below)", "Polyketide synthase gene (PKS)", "Reductase I gene (THN1)", "Scytalone dehydratase gene (SD)", "Reductase II gene (THN2)", "Oxidases"}; String defaultEdit = "Currently, no gene is dirupted"; Pathway pp, p; DrawPathway d; public PathwayView(int x){ super("Pathway Modelling and Visualization"); setSize(800, 450); addWindowListener(new WindowAdapter() { public void windowClosing(WindowEvent e) {dispose();} }); pp = new Pathway(x); init(pp); setVisible(true); }  public void init(Pathway pl){ .  Container contentPane = this.getContentPane(); Font labelFont = new Font("SanSerif, Font.BOLD, 18); p = pl; //set the container to the grid bag layout and //define the constarint obj GridBagLayout gridbag = new GridBagLayout(); contentPane. setLayout(gridbag); GridBagConstraints c = new GridBagConstraints(); //setings for constraint obj instant varaibales  191  c.fill= c.BOTH; //setting for labell c.insets = new Insets(l 0,10,10,20); c.gridx = 0; c.gridy = 0; c.gridwidth = 1; c.gridheight = 1; c.anchor = c.WEST; c.weightx = 0.0; c.weighty = 0.0; JLabel 11 = new JLabel("Fungal DHN Melanin Pathway (Modelling and Visualization)"); H.setFont(labelFont); gridbag.setConstraints(ll, c); contentPane.add(ll); //add the labell //setting for label2 c.gridx = 0; c.gridy = 1; c.gridwidth = 1; c.gridheight = 1; c.anchor = c.WEST; c.weightx = 0.0; c.weighty = 0.0; JLabel 12 = new JLabel("Gene disruption test, select a gene and get a color with:"); 12 .setFont(labelFont); gridbag.setConstraints(12, c); contenfPane.add(12); //add the label2 //add a combox, containing gene names c.gridx = 4; c.gridy = 1; c.gridwidth = 1; c.gridheight = 1; c. anchor = c. CENTER; c.weightx = 0.0; c.weighty = 0.0; JComboBox genes = new JComboBox(geneNames); genes.setOpaque(true); genes .setEditable(false); // configure the combo box editor. genes.configureEditor(genes.getEditor(), defaultEdit); // rows to be visible without scrollbars. genes.setMaximumRowCount(6); // set the combo box editor colors and font. ComboBoxEditor cboxEditor = genes.getEditor(); Component editorComp = cboxEditor.getEditorComponent(); editorComp.setBackground(Color.white); editorComp.setForeground(Color.blue); Font fl = new Font("Dialog", Font.PLAIN, 12); editorComp. setFont(fl); // font for the combo box popup list. genes. setFont(fl); //genes. addItemListener( this); genes.addActionListener(this); gridbag.setConstraints(genes, c); contentPane.add(genes); //add the drawPathway c.gridx = 0; c.gridy = 3; c.gridwidth = 5; c.gridheight =1; c.anchor = c.CENTER; c.weightx = 1.0; c.weighty = 1.0;  192  c.fill = c.BOTH; d = new DrawPathway(p); gridbag.setConstraints(d, c); contentPane.add(d); }  // public void itemStateChanged(ItemEvent e){} public void actionPerformed(ActionEvent e) { JComboBox source = (JComboBox) e.getSource(); String item = (String) source.getSelectedItem(); if (item.equals(geneNames[0])){ this, setVisible(false); new PathwayView(7); }  for (int j = 1; j < geneNames.length; j++) { if (item.equals(geneNames[j])){ this.setVisible(false); //int length; if (item.equals("Oxidases")) j=j+i; new PathwayView(j); }  } }  public static void main(String s[]) { PathwayView f = new PathwayView(7); f.show(); } }  //Helpltemjava import java.awt.*; import java.awt.event.*; /** this class shows help information*/ public class Helpltem extends Frame {  /** text Area */ private TextArea tal; /** panels */ private Panel pi, p2, p3, p4, p5; /** Constructor */ public Helpltem() { super(); // call Frame's empty constructor setTitle("Help information"); sizeandplaceWindow(); setBackground(Color.lightGray); addWindowListener(new WindowCloser()); init(); buildHM(); setVisible(true); }  193  /** this method is to instantiate variables */ public void init(){ tal = new TextArea(8, 50); tal.setFont(newFont("ARIAL", Font.BOLD, 14)); pi = newPanel(); p2 = new Panel(); p3 = new Panel(); p4 = new Panel(); p5 = new Panel(); }  /** build the view */ public void buildHM() {tal.setText(" This program contains the gene/enzyme/mutant\n" + " information about the fungal DHN-melanin pathway.\n\n" + " To search genes, click the Gene Search button.W + " To search enzymes, click the Enzyme Search button.W + " To search gene/enzyme using species as a seraching\n" + " key, click Species Search button.W ); pl.add(tal); add("North", p3); add("South", p2); add("Center", pi); add("East", p5); add("West", p4); }  /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter {public void windowClosing(WindowEvent e){ dispose(); } }  /** this private method sizes and places theframein the right upper corner of the screen private void sizeandplaceWindow() {  this.setSize(500, 200); this.setResizable(false); this.setLocation(200, 200);  }  public static void main(String args[]) { Helpltem hm = new Helpltem(); } } II  ********************************************  // MainFrame.java import Java.awt.*; import java.awt.event.*; import j a vax. swing .event. *; 194  import javax.swing.*; import javax.swing.border.*; import java.io.*; /**  * The calass is responsible for providing an interface to the services. */ public class MainFrame extends JFrame { /** the container reference */ Container c = null; /** model variable */ protected Model mymodel; /** file variable */ private File files; /**label variable */ private JLabel 1; /** a menubar in the main view */ private JMenuBar mb; /** menu items in the main view */ private JMenuItem findfile, content, about; /** menu in the main view */ private JMenu file, help; /**panel*/ private JPanel p, pi; /** buttons */ private JButton gene, enzyme, species, pathway; String s; Frame myView; /** a loadview variable */ //LoadView loadview; /* Constructor */ public MainFrame(Model m)  {  super(); mymodel = m; s = "frame"; setTitle("Gene Inquiry System"); //configure the frame using its content pane c = this.getContenfPane(); c.setBackground(Color.lightGray); sizeandplace Window(); init(); buildMenu(); setVisible(true); addWindowListener(new WindowCloser());  }  /** this method is to initialize the variables */ public void init() {  195  mb = new JMenuBar(); mb.setBorder(new BevelBorder(BevelBorder.RAISED)); mb. setB orderP ainted(true); file = new JMenu("File", true); file.setFont(new Font("ARIAL", Font.BOLD, 14)); help = new JMenu("Help"); help.setFont(new Font("APJAL", Font.BOLD, 14)); findfile = new JMenuItem ("Exit"); findfile.setFont(newFont("ARIAL", Font.BOLD, 12)); findfile.addActionListener(new Menulistener ()); content = new JMenuItem("Help info"); content.setFont(newFont("ARIAL", Font.BOLD, 12)); content.addActionListener(new Menulistener ()); about = new JMenuItem("About"); about.setFont(new Font("ARJAL", Font.BOLD, 12)); about. addActionListener(new Menulistener ()); 1 = new JLabel("Fungal Melanin Gene System", JLabel.CENTER); l.setFont(newFont("ARIAL", Font.BOLD, 28)); p = new JPanel (); pi = new JPanel(); gene = new JButton("Gene Search"); gene. addAc tionListener(mymodel); gene.setFont(new Font("ARIAL", Font.BOLD, 16)); enzyme = new JButton("Enzyme Search"); enzyme.addActionListener(mymodel); enzyme.setFont(new Font("ARIAL", Font.BOLD, 16)); species = new JButton("Species Search"); species.addActionListener(mymodel); species.setFont(newFont("ARIAL", Font.BOLD, 16)); pathway = new JButton("Pathway Display"); pathway.addActionListener(new Menulistener ()); pathway.setFont(newFont("ARIAL", Font.BOLD, 16)); }  /** build the menu structure */ public void buildMenu() {  c.add(mb, BorderLayout.NORTH); mb.add(file); file.add(findfile); pl.add(l); //menu "help" mb.add(help); help .addSeparator(); help. add(contenf); help.addSeparator(); help.add(about); //add buttons p.add(gene); p.add(enzyme); p.add(species); p.add(pathway); c.add("Center",pl); 196  c.add("South", p); }  /** this class is to implement the ActionListener interface */ private class Menulistener implements ActionListener { /** the method is listening */ public void actionPerformed(ActionEvent e){ Object source = e.getSource(); if(source == about) { AboutView av = new AboutView(myView, "About Gene System", true); av.start(); }else if (source = content)! Help Item helpitem = new Helpltem(); helpitem.setVisible(true); }else if (source == pathway)! PathwayView p = new PathwayView(7); p.setVisible(true); }else if (source == findfile) {  System.exit(O); }  }  }  /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter {  }  public void windowClosing(WindowEvent e){ System.exit(0); }  /** this private method sizes and places the frame in the middle of the screen */ private void sizeandplaceWindow() {  this.setSize(800, 450); this. setResizable(false); Dimension frameD = new Dimension(800, 450); Dimension screenD = new Dimension(); screenD = Toolkit.getDefaultToolkit().getScreenSize(); if(frameD. width >= screenD. width) this.setLocation(l, 1); this.setLocation(((screenD.width - frameD.width)/2), ((screenD.height - frameD.height)/2)); this.setSize(frameD.width, frameD.height);  }  // testing public static void main(String arg[]){ Model a = new Model(); MainFrame mf = new MainFrame(a); }  197  } II  *************************************************  // SpeciesView.java import javax.swing. *; import java.awt.*; import java.awt.event.*; import javax.swing.border.*; /**  * The calass is responsible for providing an interface for species search. */ public class SpeciesView extends JFrame { /** declare the references. */ JComboBox cbox = null; JComboBox cboxl = null; Container c = null; /** define an array of species name to be shown in the * combo box pull-down list, by default. */ String[] defaultSpecies = { "Ophiostoma floccosum", "Magnaporthe grisea", "Colletotrichum lagenarium"}; String defaultEdit = "Ophiostoma floccosum"; /** define an array of either gene or enzyme to be shown in the * combo box pull-down list, by default. */ String[] defaultGen = { "Gene", "Enzyme"}; String defaultEditl = "Gene"; /** model variable */ protected Model mymodel; /** constructor */ public SpeciesView (Model m) {  super(); setTitle("Species Search View"); mymodel = m; //configure the frame using its content pane c = this.getContenfPane(); c. setB ackground(Color. lightGray); c.setLayout(new GridLayout(l,l)); init(); sizeandplace Window(); addWindowListener(new WindowCloser()); setDefaultCloseOperation(WindowConstants.DISPOSE_ON_CLOSE);  }  /** construct the view */ public void init() { // prepare the container with layout 198  JPanel pi = new JPanel(); p 1 .setLayout(new BorderLayout()); // set the title border around the panel. TitledBorder borderl = new TitledBorder( new LineBorder(Color.blue), "It will list all genes or enzymes for the selected species"); border 1. setTitleColor(Color.blue); p 1. setBorder(border 1); JPanel p3 = new JPanel(); p3 .setLayout(new GridLayout( 1,2)); JPanel p4 = new JPanel(); p4.setLayout(new GridLayout(l,3)); JPanel p7 = new JPanel(); // create the combo box with the list of default sites. cbox = new JComboBox(defaultSpecies); cbox.setOpaque(true); cboxl = new JComboBox(defaultGen); cbox 1. setOpaque(true); // make the combo box ineditable. cbox.setEditable(false); cbox 1. setEditable(false); // configure the combo box editor. cbox.configureEditor(cbox.getEditor(), defaultEdit); cboxl.configureEditor(cbox.gefEditor(), defaultEdit 1); // rows to be visible without scrollbars. cbox.sefMaximumRowCount(3); cboxl .setMaximumRowCount(2); // set the combo box editor colors and font. ComboBoxEditor cboxEditor = cbox.getEditor(); Component editorComp = cboxEditor.getEditorComponent(); editorComp.setBackground(Color.white); editorComp.setForeground(Color.blue); Font fl = new Font("Dialog", Font.PLAIN, 14); editorComp. setFont(fl); ComboBoxEditor cboxEditorl = cboxl.getEditor(); Component editorComp 1 = cboxEditorl.getEditorComponent(); editorComp 1 .setBackground(Color.white); editorComp 1. setForeground(Color.blue); Font f2 = new Font("Dialog", Font.PLAIN, 14); editorComp 1 .setFont(f2); // font for the combo box popup list. cbox.setFont(fl); cboxl .setFont(f2); // label to indicate what to do. JLabel labell = new JLabel("Select fungal species:", JLabel.CENTER); 199  labell.setFont(newFont("SansSerif, Font.BOLD, 16)); JLabel label2 = new JLabel("Select gene/enzyme:", JLabel.CENTER); label2.setFont(newFont("SansSerif', Font.BOLD, 16)); // button for search JButton bl = new JButton("Seek"); bl.setFont(newFont("SansSerif, Font.BOLD, 16)); bl .addActionListener(mymodel); //construct thefirstsearch block p3.add(labell); p3.add(cbox); p4.add(label2); p4.add(cboxl); p7.add(bl); pl.add(p7, BorderLayout.SOUTH); pl.add(p3, BorderLayout.CENTER); pl.add(p4, BorderLayout.NORTH); //add the blocks to the frame c.add(pl); }  /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter { public void windowClosing(WindowEvent e){ dispose(); } }  /** this private method sizes and places theframein the middle of the screen */ private void sizeandplaceWindow()  {  }  this.setSize(400, 150); this.setResizable(false); DimensionframeD= new Dimension(400, 150); Dimension screenD = new Dimension(); screenD = Toolkit.getDefaultToolkit().getScreenSize(); if(frameD. width >= screenD.width) this.setLocation(l, 1); this.setLocation(((screenD.width - frameD.width)/2), ((screenD.height - frameD.height)/2)); this.setSize(frameD.width, frameD.height);  //testing public static void main(String arg[]) { Model a = new Model(); SpeciesView gv = new SpeciesView(a); }  }  200  II  *******************************************  //AboutView.j ava // This Class will display "About" window and play animation in a thread // Toolkit has been used to fetch the images and loading them in array // It is invoked, when menu option About is clicked import java.awt.*; import java.awt.event.*; import java.util.*; import java.awt.Toolkit; public class AboutView extends Thread implements Runnable, ActionListener, WindowListener {  //  Dialog d; Frame d; Button OK; Panel pi, p2, plabel; Label heading,ll, 12,13,14,15,16; String gl= "", g2= "", g3= "", g4= "", g5= " g 6 = ""; Image images[]; int totallmages = 4, // total number of images currentlmage = 0, // current image subscript sleepTime = 500; // milliseconds to sleep String imageName = "duke"; // base name of images Thread animate; // animation thread Graphics gContext; int height = 0, width = 0; AboutViewCanvas canvas; public AboutView(Frame parent, String s, boolean bvalue) {  d = new Frame(s); d.setSize(400, 225); d.setLocation(200,150); d.setBackground(Color.lightGray); d.setResizable(false); init(); startanimation();  public void init() { loadImages(); addGUI(); canvas = new AboutViewCanvas(); d.add("Norfh", pi); d.add("Center", canvas); d.add("South", p2); // add the listeners OK.addActionListener(this); d. addWindo wListener(this); d.setVisible(true); 201  }  public void loadImages() { images = new Imagef totallmages ]; for (int i = 0; i < images.length; i++) images[ i ] = Toolkit.getDefaultToolkit().getImage(imageName + i + ".gif') }  // instantiate the Button, Label & Panel for our Dialog box public void addGUI(){ pi = new Panel(); p 1 .setLayout(new BorderLayout()); p 1 .setBackground(Color.lightGray); heading = new Label("Fungal Melanin Gene System", Label.CENTER); heading.setFont(new Font ("Sans Serif, Font.ITALIC, 18)); heading.sefForeground(Color .blue); gl = "Fungal Melanin Gene System, version 1.0.0" ; g3 =" All Rights Reserved "; 11 = new Label(gl, Label.CENTER); 11 .setflackground(Color.lightGray); 11. setFont(new Font ("ARIAL", Font.BOLD, 14)); 12 = new Label(g2, Label.CENTER); 12. setB ackground(Color. lightGray); 12. setFont(new Font ("ARIAL", Font.BOLD, 14)); 13 = new Label(g3, Label.CENTER); 13 .setBackground(Color.lightGray); 13. setFont(new Font ("ARIAL", Font.BOLD, 14)); plabel = new Panel(); plabel.add(ll); p2 = new Panel(); p2.add(OK = new Button("OK")); // now add the components to the frame pi.add("North", heading); pi.add("Center", plabel); pl.add("South", 13); }  public Dimension getPreferredSize(){ return new Dimension(width, height);} public Dimension gefMinimumSize(){ return getPreferredSize();} // start the applet public void startanimation() {  //always start with 1st image currentlmage = 0; // create a new animation thread when user visits page if ( animate = null) { 202  animate = new Thread( this ); animate. start(); }  }  // override update to eliminate flicker public void run() {  while (true){ canvas.setPic(images[currentImage]); currentlmage = ( currentlmage + 1) % totallmages; try{ Thread. sleep( sleepTime); }  catch (InterruptedException e) {} } }  /** since we are handling the event processing, do so */ public void actionPerformed(ActionEvent e) {  // check the cmd field to see if it's our button if(e.getActionCommand() = "OK") {  }  }  d.dispose(); animate = null;  public void windowClosing(WindowEvent e){ //animate.stop(); animate = null; d.hide(); d.dispose(); //animate.destroyO; }  public void windowActivated(WindowEvent e){} public void windowClosed(WindowEvent e){} public void windowDeiconified(WindowEvent e){} public void windowIconified(WindowEvent e){} public void windowOpened(WindowEvent e){} public void windowDeactivated(WindowEvent e){}  II  ***************************************************  // AboutVie wCanvas Java // This class is used to create a canvas for About Class inorder to draw animated object in it import java.awt.*; public class AboutViewCanvas extends Canvas { Image image; 203  public AboutViewCanvas () {} public void setPic( Image imagefile ) { image = imagefile; repaint();  } public void paint( Graphics g) {  g.drawlmage(image, 150, 10, this ); }  } II ******************************************  //Display.java import javax.swing.*; import java.awt.event.*; import java.awt.*; /** this class displays information*/ public class Display extends JFrame { // Declare the references for the following objects. Container container; JLabel label = null; JTextArea textArea = null; JButton insertButton = null; JButton fastaButton = null; JButton cutButton = null; JButton copyButton = null; JButton pasteButton = null; JButton xmlButton = null; /** Constructor */ public Display() { super("Display information"); sizeandplaceWindow(); addWindowListener(new WindowCloser()); init(); setVisible(true); }  public void init () { // Assign a name to the frame and obtain a handle // on the frame's content pane, container = this.getContentPane(); // Create the fonts for label and text area. 204  Font labelFont = new Font("SanSerif', Font.BOLD, 14); Font textFont = new Font("Dialog", Font.PLAIN, 12); // Use the gridbag layout for the applet. GridBagLayout gridbag = new GridBagLayout(); container.setLayout(gridbag); GridBagConstraints c = new GridBagConstraints(); c. insets = new Insets(2,10,10,2); // Add the xml button, c.gridx = 0; c.gridy = 0; c.gridwidth = 1; c.gridheight =1; c.fill = c.BOTH; xmlButton = new JButton("Export XML"); xmlButton.setBackground(Color.lightGray); gridbag. setConstraints(xmlButton, c); container.add(xmlButton); // add the paste button ButtonListener xmlButtonListener = new ButtonListener(); xmlButton. addActionListener(xmlButtonListener); // Add the Delete button. c.gridx = 1; c.gridy = 0; fastaButton = new JButton("Fasta File"); fastaButton.setBackground(Color.lightGray); gridbag.setConstraints(fastaButton, c); container.add(fastaButton); // add the delete button ButtonListener dlButtonListener = new ButtonListener(); fastaButton.addActionListener(dlButtonListener); // Add the Cut button, c.gridx = 2; c.gridy = 0; cutButton = new JButton("Cut"); cutButton.setBackground(Color.lightGray); gridbag.setConstraints(cutButton, c); container.add(cutButton); // add the cut button ButtonListener ctButtonListener = new ButtonListener(); cutButton.addActionListener(ctButtonListener); // Add the Copy button, c.gridx = 3; c.gridy = 0; copyButton = new JButton("Copy"); copyButton.setBackground(Color.lightGray); gridbag.setConstraints(copyButton, c); container.add(copyButton); // add the copy button ButtonListener cpButtonListener = new ButtonListener(); copyButton.addActionListener(cpButtonListener); // Add the Paste button, c.gridx = 4; c.gridy = 0; pasteButton = new JButton("Paste"); pasteButton.setBackground(Color.lightGray); gridbag. setConstraints(pasteButton, c); container.add(pasteButton); // add the paste button ButtonListener psButtonListener = new ButtonListener(); pasteButton.addActionListener(psBurtonListener);  205  // Add the text area, c.gridx = 0; c.gridy = 1; c.gridwidth = 5; c.gridheight = 1; c.weightx = 1.0; c.weighty = 1.0; c.anchor = c.CENTER; c.fill = c.BOTH; textArea = new JTextArea(10, // Number of rows. 30); // Number of columns. JScrollPane sp = new JScrollPane(textArea); textArea.setFont(textFont); textArea.setBackground(Color.white); textArea.setSelectionColor(Color. yellow); textArea.setTabSize(5); // The tab size. textArea.setLineWrap(true); // Wrap the line at the container end. gridbag.setConstraints(sp, c); container.add(sp); // Add the text area. // Add the window listener to close the frame // and display it with the specified size. setDefaultCloseOperation(WindowConstants.DISPOSEONCLOSE); }  /** this inner class deals with closing the window */ private class WindowCloser extends WindowAdapter { public void windowClosing(WindowEvent e){ dispose(); }  } //Button listener class. class ButtonListener implements ActionListener { public void actionPerformed(ActionEvent e) { JButton b = (JButton) e.getSource(); if(b = fastaButton) { ; // Fasta file will be created }  else if (b = cutButton) { textArea.cut(); // Cut operation. }  else if (b = copyButton) { textArea.copyO; // Copy operation. }  else if (b = pasteButton) { textArea.paste(); // Paste operation. }  else if (b = xmlButton) { ; // export xml file }  206  } }  /** this private method sizes and places the frame in the right upper corner of the screen */ private void sizeandplaceWindow() {  this.setSize(450, 500); this. setResizable(false); this.setLocation( 100, 50);  }  public static void main(String args[]){ Display hm = new Display(); } } II  ***********************************************  // Phenotype Java /** this class defines the information of a pheno type, * wild type and mutant which is defective in some gene */ public class Phenotype {  /** the defective gene */ private Gene g; /** the phenotype description */ private String description; /** the accumulated compound */ private String accumulator; /** species name */ private String speciesName; /** a constructor */ public Phenotype() { description = "Unknown"; accumulator = "Unknown"; }  /** a convenience constructor */ public Phenotype(String i, String sn) {  description = i; accumulator = sn;  } /** the method is to set the description */ public void setDescription (String d) { description = d; }  /** the method is to set accumulator */ 207  public void setAccumulator(Enzyme e) { accumulator = e.getSubName(); }  /** the method is to set accumulator */ public void setAccumulator(String e) I  accumulator = e; }  /** the method is to obtain the description */ public String getDescription() { }  return description;  /** the method is to obtain the accumulator */ public String getAccumulator() {  return accumulator; } } II  ******************************************  //Version: 1.0.0 //  // ©Author: Edwin (Honglong) Wang //  // Tested on Java(TM) 2 Platform //  // Sun Microsystems, Inc., // 901 San Antonio Road, // Palo Alto, CA 94303 USA. //  // Copyright (c) 2000 // E. Wang // University of British Columbia, // Vancouver, Canada //  // All Rights Reserved //  // License: non-commercial II  ********************************************  // Model.java import javax.swing.event.*; import j avax. swing. *; import java.util.*; import java.awt.*; import java.lang.*; import java.io.*; import java.awt.event.*;  208  /** the Model class, provides all methods to processe data, trigger several major views */ public class Model implements ActionListener {  /** Gene list variable */ private static Genelist myGenelist; /** Enzymelist variable */ private static Enzymelist myEnzymelist; /** collection of rnzymes */ private ArrayList collection; //views will be triggered by Model protected Gene View geneview; protected Enzyme View enzyview; protected SpeciesView speciesview; protected MainFrame mf; /** HashMap contains gene-mutant pair */ protected HashMap myHM; /** enzyme names */ String [] enzyme_name = {"Polyketide synthase", "Reductase", "Scytalone dehydratase"}; /** species names */ Stringf] species_name = {"Ophiostoma floccosum", "Magnaporthe grisea", "Colletotrichum lagenarium"}; /** the constructor of the model class */ public Model() {  //HashMap contains gene as key, phenotype as value myHM = new HashMap(); collection = new ArrayList(); geneview = new GeneView(this); enzyview = new EnzymeView(this); speciesview = new SpeciesView(this); mf = new MainFrame(this); fhis.setupDataO;  }  /** This methos is to set up the databases for gene, enzyme, and phenotype */ public void setupData() {  synthase",  //data String[] gene_name= {"OSD1", "THN1", "THN2", "mgSD", "T4HRgene", "T3HR gene", "PKS1", "SCD1", "THR1"}; String[] e_name = {"Scytalone dehydratase", "Reductase", "Reductase", "Scytalone dehydratase", "Reductase", "Reductase","Polyketide "Scytalone dehydratase", "Reductase"}; StringO enzyme_id = {"SD", "THN1", "THN2", "SD", "THN1", "THN2", "PKS", "THN2"}; for (int i = 0; i < species_name.length; i++) 209  for(intj = 0;j<3;j++) { int c = 0; Gene gc = new Gene(); c = i*3+j; gc.setSpeciesName(species_name[i]); gc. setName(gene_name [c]); //open the file and read sequences // buffered input file try{ BufferedReader in = new BufferedReader( new FileReader(("gene" + + ".txt"))); c  space,  String s, s2 = new String(); while((s = in.readLine())!= null) s2 += s.toUpperCase().trim(); // change to upper case, trim gc.setSeq(s2); in.close();  // and cancat;  } catch(FileNotFoundException e) { System.out.println( "File Not Found:" + "gene" + c + ".txt"); } catch(IOException e) { System.out.println("IO Exception"); }  Enzyme ec = new Enzyme(); ec.setSpeciesName(species_name[i]); ec. setName(e_name [c]); ec.setID(enzyme_id[c]); ec.setGene(gc); //set the substrate and reaction product names if(ec.getID().equals("SD")) {  ec. setSubName(" Scytalone"); ec.setProductName("l, 3, 8-THN"); }else if(ec.getID().equals("THNl")) { ec.setSubName("l, 3, 6, 8-THN"); ec.setProductName("Scytalone"); }else if(ec.getID().equals("THN2")) { ec.setSubNameC'l, 3, 8-THN"); ec.setProductName("Vermelone"); }else if (ec.getID().equals("PKS")) { ec.setSubName("Acetate"); ec.setProductName("l, 3, 6, 8-THN"); }  //open the file and read sequences // buffered input file try{ BufferedReader in = 210  new BufferedReader( new FileReader(("enzy" + c + ".txt"))); String si, s21 = new String(); while((sl = in.readLine())!= null) s21 += sl.toUpperCase().trim(); // change to upper case, trim space, // and cancat ec.setSeq(s21); in.close(); } catch(FileNotFoundException e) { System.out.println( "File Not Found:" + "enzy" + c + ".txt"); } catch(IOException e) { System.out.println("ip Exception"); //add to the list collection, add(ec); //set up my HashMap Phenotype pc = new Phenotype(); if(gc.getName().equals(gene_name[0]) || gc.getName().equals(gene_name[l]) || gc.getName(). equals(gene_name [2 ])) else if (gc.getName().equals(gene_name[3]) || gc.getName().equals(gene_name[7])) {  pc.setDescription("Reddish color"); pc .setAccumulator(ec); }else if (gc.getName().equals(gene_name[5]) || gc.getName().equals(gene_name[8])) { pc.setDescription("Brown color"); pc.setAccumulator(ec); }else if (gc.getName().equals(gene_name[4]))  { pc.setDescription("Black color"); pc.setAccumulator("Melanin"); }else  { pc.setDescription("Albino color"); pc. setAccumulator(ec);  }  myHM.put(gc, pc);  } } /** this method is display enzyme information */ public String enzyDisplay(Enzyme tempEnzy) { return ("\n Enzyme Name: " + tempEnzy.getName() + " , " + tempEnzy.getID() + "\n Species Name: " + tempEnzy.getSpeciesName() + "\n Substrate: " + tempEnzy.getSubName() + "\n Reaction product: " +  211  tempEnzy.getProductName() + "\n Protein sequence: \n>" + tempEnzy.getName() + " [" + tempEnzy.getSpeciesName() + "]\n" + tempEnzy.getSeqO + "\n" + "*******************" + "\n");  } /** this method is display enzyme information */ public String geneDisplay(Enzyme tempEnzy) { String si, s2 = null; Phenotype p = null; p = (Phenotype) myHM.get(tempEnzy.getGene()); si = p.getDescription(); s2 = p.getAccumulator(); return ("\n Gene Name: " + tempEnzy.getGene().getName() + "\n Its enzyme Name: " + tempEnzy.getName() + "\n Species Name: " + tempEnzy.getSpeciesName() + "\n Gene mutant information:\n Mutant color: " + si + "\n Compound accumulated in the mutant's melanin pathway: + s2 + "\n Gene sequence: \n> " + tempEnzy.getName() + " [" + tempEnzy.getSpeciesName() + "]\n" + tempEnzy.getGene().getSeq() + "\n" + +  /** the method is listening */ public void actionPerformed(ActionEvent e) {if (e.getActionCommand() = "Gene Search") geneview.setVisible(true); else if (e.getActionCommand() = "Enzyme Search") {enzyview. setVisible(true); }else if (e.getActionCommandO = "Species Search") { speciesview.setVisible(true); }else if (e.getActionCommandO = "Find"){ if(geneview.t 1 .getText().equals("")) geneview.tl.setText("please enter data."); else{Display d = new Display(); d.textArea.append("Nothing matches your search, please check and search again\n"); d.textArea.append("The key words for searching are: Scytalone dehydratase, Reductase, or Polyketide synthase"); Enzyme tempEnzy = null; for(int i = 0; i < enzyme_name.length; i++){ if (geneview.tl .getText().equalsIgnoreCase(enzyme_name[i])) { for(int j = 0; j < species_name.length; j++){ if (geneview.cbox.getSelectedItem().toString().equalsIgnoreCase(species_name[j])){ d.textArea.setTextC'");  212  for (int v = 0; v < collection.size(); v++) { tempEnzy = (Enzyme) collection.get(v); if(tempEnzy.getSpeciesName().equals(species_name[j]) && tempEnzy.getName().equals(enzyme_name[i])){ d.textArea.append(geneDisplay(tempEnzy)); } }  } } } }  }  }else if (e.getActionCommandO == "Go"){ if(geneview.t2.getText().equals("")) geneview.t2.setText("please enter data."); else{ Display d = new Display(); d.textArea.append("Nothing matches your search, please check and search again\n"); d.textArea.append("The key words for searching are: Scytalone dehydratase, Reductase, or Polyketide synthase"); Enzyme tempEnzy = null; for(int i = 0; i < enzyme_name.length; i++){ if (geneview.t2.getText().equalsIgnoreCase(enzyme_name[i])) { d.textArea.setText(""); for (int v = 0; v < collection.size(); v++){ tempEnzy = (Enzyme) collection.get(v); if(tempEnzy.getName().equals(enzyme_name[i])){ d.textArea.append(geneDisplay( tempEnzy)); }  } } }  } }else if (e.getActionCommandO = "Search"){ if(enzyview.tl.getText().equals("")) enzyview.tl.setText("please enter data."); else{ Display d = new Display(); d.textArea.append("Nothing matches your search, please check and search again\n"); d.textArea.append("The key words for searching are: Scytalone dehydratase, Reductase, or Polyketide synthase"); Enzyme tempEnzy = null; for(int i = 0; i < enzyme_name.length; i++){ if (enzyview.tl .getText().equalsIgnoreCase(enzyme_name[i])) { d.textArea.setText(""); for(int j = 0; j < species_name.length; j++){ if (enzyview.cbox.getSelectedItem().toString().equalsIgnoreCase(species_name[j])){ for (int v = 0; v < collection.size(); v++){ tempEnzy = (Enzyme) collection.get(v); if(tempEnzy.getSpeciesName().equals(species_name[j]) && tempEnzy.getName().equals(enzyme_name[i])){  213  d.textArea.append(enzyDisplay( tempEnzy)); } }  } } } } }  search again\n");  }else if (e.getActionCommandO = "Hunt"){ if(enzyvie w. t2. ge tText(). equals("")) enzyview.t2.setText("please enter data."); else{ Display d = new Display(); d.textArea.append("Nothing matches your search, please check and d.textArea.append("The key words for searching are: Scytalone dehydratase, Reductase, or Polyketide synthase"); Enzyme tempEnzy = null; for(int i = 0; i < enzymename. length; i++){ if (enzyview.t2.getText().equalsIgnoreCase(enzyme_name[i])) { d.textArea.setText(""); for (int v = 0; v < collection.size(); v++){ tempEnzy = (Enzyme) collection.get(v);  if(tempEnzy.getName().equals(enzyme_name[i])){ d.textArea.append(enzyDisplay( tempEnzy)); } }  }  }  }  }else if (e.getActionCommandO = "Seek"){ Display d = new Display(); Enzyme tempEnzy = null; for(int j = 0; j < species_name.length; j++){ if (speciesview.cbox.getSelectedItem().toString0.equals(species_name|j])){ if (speciesview.cboxl.getSelectedItem0.toString().equals("Enzyme")){ for (int v = 0; v < collection.size(); v++){ tempEnzy = (Enzyme) collection.get(v); if(tempEnzy.getSpeciesName().equals(species_name[j])) d.textArea.append(enzyDisplay(tempEnzy)); }  }else{  for (int v = 0; v < collection.size(); v++) { tempEnzy = (Enzyme) collection.get(v); if(tempEnzy.getSpeciesName().equals(species_name[j])) d.textArea.append(geneDisplay(tempEnzy)); } } } }  214  }  } /** THIS IS T H E STRAT POINT OF T H E PROGRAM  public static void main(Struig[] args) { Model m = new Model (); } }  215  

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