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Molecular analysis and physiological roles of subtilases in sapstaining fungi Hoffman, Bradford Glenn 2003

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M O L E C U L A R A N A L Y S I S A N D P H Y S I O L O G I C A L R O L E S O F S U B T I L A S E S I N S A P S T A I N I N G F U N G I by B R A D F O R D G L E N N H O F F M A N B.Sc , The University of British Columbia, 1996 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S Department of Wood Science We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A J U N E , 2003 © Bradford Glenn Hoffman, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Abstract The forest products industry is the largest industry in British Columbia and one of the main industries in Canada. Sapstain fungi cause cosmetic defects in wood and are a major problem to the industry. Millions of dollars are spent each year to prevent the growth of these fungi in sawn wood; however, current control methods are unsatisfactory. Modern chemical treatments have raised environmental concerns and do not have the duration of effectiveness desired by the industry. Kiln drying is expensive and not always practical and the rewetting of wood during transport can allow subsequent fungal growth and stain production. Current biological control agents are also unsatisfactory as they are unreliable and do not grow well in many environments. In order to develop new methods for stain control it is vital to fully understand the relationship between the stairiing fungi and the host wood. It is likely that the best stain control strategies will integrate these new methods with existing techniques, incorporating the strengths of each. The nutrient content in wood affects the growth and melanin production of sapstaining fungi, and wood extractives are a major source of nutrients for these fungi. Levels of easily assimilated nitrogen sources in wood are extremely low and organic sources of nitrogen, such as proteins, are vital for the growth of sapstaining fungi. Fungi produce extracellular proteases in order to utilise these nitrogen sources, and these enzymes are likely necessary for fungal growth on wood. The objective of this thesis was to understand the role of subtilases in the physiology of sapstaining fungi. To achieve this objective several steps were taken. First, a comprehensive I l l -study of the distribution and sequence variation of subtilases in sapstaining fungi was carried out. Using the obtained sequences three groups of fungal subtilases were delineated. This is the Erst report of these groups. Seven full-length sequences were also obtained providing a basis for future work on these genes. Second, the regulation of three representative subtilase genes was detemiined, extending what is known about the regulation of fungal subtilase genes and providing important information on the potential roles of these genes. Finally, the targeted disruption of opill is described. This is the first targeted disruption in any sapstaining species of Ophiostoma. The analysis of the obtained disruptants clearly showed that Opill is involved in nutrient acquisition for exogenous protein sources during growth in wood. This suggests that Opill could be a useful target for the development of new control strategies. This work also demonstrates the importance of nitrogen in fungal growth on wood, which could lead to better decisions in tree harvesting and be used to assist in the improved application of existing control strategies. - iv -Table of Contents Page Abstract ii Table of Contents iv List of Tables ix List of Figures x List of Abbreviations xiii Acknowledgements xvii Chapter 1: General Introduction and Research Objectives 1 /. /. The Problem of Sapstain to the Forest Products Industry 1 1.2. Sapstaining fungi 4 1.2.1. Clas sification o f Stairiing Fungi 4 1.2.2. Biology of Sapstaining Fungi 6 1.2.3. Growth of Sapstaining Fungi in Wood 9 1.3. Regulation of Nitrogen and Carbon Catabolic Pathways 13 1.3.1. Global Nitrogen Repression and Activation 13 1.3.2. Regulation of Carbon Catabolic Pathways 15 1.4. Fungalproteases 16 1.4.1. Proteases 16 - V -1.4.2. Serine Proteases 17 1.4.3. Subtilases 22 1.4.4. Fungal Subtilases 22 1.4.5. Regulation of Fungal Protease Genes 28 /. 5. Research approach and objectives 32 Chapter 2 : Cloning and genetic analysis of subtilases in sapstaining fungi 34 2.1. Introduction 34 2.2. Materials and Methods 36 2.2.1. Fungal and bacterial strains 36 2.2.2. Fungal and bacterial growth conditions in culture 37 2.2.3. Purification of Genomic D N A and Total R N A from Fungal Strains 37 2.2.4. Purification of plasmids from bacterial strains 38 2.2.5. Purification of X phage D N A 38 2.2.6. Polymerase Chain Reactions (PCR) and Randomly Amplified c D N A Ends-Polymerase Chain Reactions (RACE-PCR) 39 2.2.7. P C R - R F L P analysis 41 2.2.8. Vertical Ge l Electrophoresis and Purification of D N A from agarose gels ...41 2.2.9. Cloning and sequencing of subtilase gene fragments 42 2.2.10. Dot blotting and Southern analysis 42 2.2.11. Screening of O.floccosum 387N X library 44 2.2.12. Hybridisations and Chemuuminescent Signal Detection 44 2.3. Results 46 - v i -2.3.1. PCR screening and dot blot analysis 46 2.3.2. PCR-RFLP analysis 49 2.3.3. Sequence analysis of PR04/PR07 PCR products 51 2.3.4. Screening of an O.floccosum 387N genomic A. library 54 2.3.5. RACE-PCR 54 2.3.6. Analysis of full-length sequences 58 2.3.7. Southern blot analysis 65 2.4. Discussion 68 2.5. Conclusions 72 Chapter 3 : Regulational analysis and heterologous expression of subtilase genes from sapstaining fungi 73 3.1. Introduction 73 3.2. Materials and Methods 75 3.2.1. Fungal and Bacterial strains and growth conditions 75 3.2.2. Protease assays 75 3.2.3. Purification of Genomic D N A , Total RNA and mRNA 77 3.2.4. Multiplex RT-PCR 78 3.2.5. Northern Analysis 79 3.2.6. Heterologous Expression 80 3.3. Results 82 3.3.1. Nutrient Regulation of the Expression of Representative Subtilase Genes ..82 3.3.2. pH Regulation of the Expression of Representative Subtilase Genes 86 - V l l -3.3.3. The Effect of Fungal Culture Age on the Expression of Representative Subtilase Genes 88 3.3.4. Heterologous Expression 93 3.4. Discussion 98 3.5. Conclusions 103 Chapter 4 : The physiological roles of opilland opi'cin sapstaining fungi 105 4.1. Introduction 105 4.2. Materials and Methods 108 4.2.1. Fungal and Bacterial strains and growth conditions 108 4.2.2. Ergosterol Analysis 108 4.2.3. Disruption Vector construction 110 4.2.4. A. tumefaciens-medi&ted transformation 112 4.2.5. Purification of Genomic D N A and Total RNA (TRNA) 113 4.2.6. Southern Analyses 113 4.2.7. Multiplex PCR and RT-PCR 113 4.2.8. Protease assays 114 4.3. Results 115 4.3.1. Transformation of O. piceae and O. piliferum 115 4.3.2. Phenotypes of mutants generated on M E A and SMA 115 4.3.3. Southern Blot Analysis and Mutliplex PCR 119 4.3.4. Multiplex RT-PCR 124 4.3.5. Protease Assays 127 - v i i i -4.3.6. Growth on wood 129 4.4. Discussion 132 4.5. Conclusions 138 Chapter 5 : Conclusions and Future work 139 References 144 Appendix 1: Lipase screening 159 - ix -List of Tables Table 2-1: List of synthetic oligonucleotides 40 Table 2-2: Analysis of obtained full-length sequences 60 Table 3-1: Components of artificial media used in multiplex RT-PCR and northern analyses 76 Table 3-2: List of synthetic oligonucleotides 79 - X -List of Figures Figure 1-1: Example of sapstain in Pinus contorta a 2x4 and a log in cross section 2 Figure 1-2: Reactions involved in the utilization of ammonia acquired from amino acids.. 12 Figure 1-3: A schematic representation of catalytic triad of a serine protease, showing the hydrogen bonding that occurs between the members of the triad 18 Figure 1-4: A schematic representation of the 3D structure of a generic subtilase, showing a-helices as cylinders, and (3-sheets as arrows 20 Figure 1-5: A schematic representation of the catalytic mechanism of a serine protease cleaving the scissile bond of a peptide 21 Figure 1-6: A ribbon schematic representation of the 3D structure of protease K, showing the locations of the catalytic triad 24 Figure 2-1: PCR analysis results using the primers PR07 and PR04 on genomic D N A from 31 different fungal species 47 Figure 2-2: A dot blot of 31 isolates of sapstaining fungi using the Prot6 Probe 48 Figure 2-3: Taq I generated restriction digestion results, of PCR fragments generated with primers PR07 and PR04 from genomic D N A of 27 fungal isolates 50 Figure 2-4: An unrooted dendogram representation of the alignment of the inferred amino acid sequences of the PR07/PR04 amplicon sequences obtained with the homologous regions from other known fungal subtilases 52 Figure 2-5: Primary and secondary library blots hybridised with the Prot6 probe 55 Figure 2-6: PCR and PCR-RFLP analysis of nine X phage clones that hybridised to the Prot6 probe 56 Figure 2-7: Genomic D N A from a X phage clone that hybridised to the Prot6 digested with different restriction enzymes 57 Figure 2-8: R A C E PCR results of selected isolates of stairiing fungi using gene specific primers and universal primer rnix 59 Figure 2-9: Alignment of the full-length subtilase sequences obtained 61 Figure 2-10: An unrooted dendogram representation of the alignment of the inferred arnino acid sequences of the full-length sequences from other known fungal subtilases 63 - xi -Figure 2-11: An unrooted dendogram representation of the alignment of the inferred amino acid sequences of the PRE-PRO regions of subtilase sequences 64 Figure 2-12: Southern blots of genomic D N A of sapstaining fungi 66 Figure 3-1: Analysis of the regulation of the opill, opil2 and opic genes by exogenous nutrient sources 83 Figure 3-2: Extracellular proteolytic activity in response to different nutrients 85 Figure 3-3: Analysis of the regulation of the opill, opil2 and opic genes by exogenous pH. 87 Figure 3-4: Extracellular proteolytic activity in response to exogenous pH 89 Figure 3-5: Analysis of the regulation of the opill, opil2 and opic genes by fungal culture age. 90 Figure 3-6: Extracellular proteolytic activity and growth in response to culture age 92 Figure 3-7: SDS-PAGE analysis of insoluble protein from E. coli cultures induced with an increasing concentration of arabinose 94 Figure 3-8: Representative purification profile of the OpU2-tJiioredoxin fusion protein.... 95 Figure 3-9: SDS-PAGE analysis of different fractions obtained from the purification of Opil l , Opil2, and Opic 96 Figure 4-1: The construction of the disruption vector pCwPl-HPH I l l Figure 4-2: Wild type 0. piliferum (WT) and 6 selected transformants grown on M E A for two weeks 117 Figure 4-3: Wild type O. piliferum (WT) and 6 selected transformants grown on SMA for two weeks 118 Figure 4-4: Schematic representation of the wild type opill gene, a hypothetical disrupted opill gene, with the hygromycin expression cassette inserted into the gene, and a randomly integrated disruption cassette 120 Figure 4-5: Southern blot analysis of 6 selected transformants and the wild type 0. piliferum with an opill probe 121 Figure 4-6: Multiplex PCR using opill specific and ITS specific primer pairs with the genomic D N A of 6 selected transformants and wild type O. piliferum 123 Figure 4-7: PCR with the primer pair wPl - l / rP l -2 on the genomic D N A of 6 selected transformants and wild type O. piliferum 125 - X l l -Figure 4-8: Multiplex RT-PCR using opiH specific and ITS specific primer pairs with the cDNA of 6 selected transformants and wild type O.piliferum 126 Figure 4-9: Growth and proteolytic activities of wild type 0. piliferum and 6 selected transformants 128 Figure 4-10: Wild type O. piliferum (WT) and 6 selected transformants grown on lodge pole pine blocks for two weeks 130 Figure 4-11: Fungal biomass as determined by ergosterol analysis of wood infected with wild type O. piliferum and selected transformants 131 - X l l l -List of Abbreviations Numbers and Symbols 3HNR 1,3,8-teihyckoxynaphthalene reductase 4HNR 1,3,6,8-tetrahydroxynaphthalene reductase uL microlitre X lamda phage A t o C A adenine a.a. amino acid A / A l a alanine ara arabinose ATMT Agrobacterium tumefaciens mediated transformation ATP adenosine triphosphate BLAST basic local alignment search tool BLASTN standard nucleotide-nucleotide BLAST BLASTX nucleotide query - protein database BLAST bp base pair BSA bovine serum albumin C degrees Celsius C cystiene C cytosine cDNA complementary deoxyribonucleic acid cm centimetre coA coenzyme A D to F d day D aspartic acid DFP diisopropylphosphorofluoridate D H N 1,8-dihydroxynaphthalene DMSO dimethyl sulfoxide - xiv -D N A deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate E D T A eAylenediaminetetraacetic acid EtBr ethidium bromide F P L C fast performance liquid chromatograp G to K g gram G glycine G guanine H histidine H P H hygromycin B phosphotransferase hr hour Hyg hygromycin B IM induction media ITS Internal transcribed spacer K b kilobases kDa kilodaltons kg kilogram L to N L litre L / L e u leucine L B Luria-Bertani medium m meter M molar M mediionine M13F pCR2.1 plasmid insert forward primer M13R pCR2.1 plasmid insert reverse primer M E malt extract M E A malt extract agar mg milligram min minute mL m i l l i l i t r p - X V -M M minimal media mm millimetre mM milHimolar mRNA messenger ribonucleic acid Mw molecular weight N any nucleotide N normal N A D + Nicotinamide adenine dinucleotide n g nanograms nm nanometers O t o R OD optical density ORF open reading frame P proline P A G E polyacrylamide gel electrophoresis PCR polymerase chain reaction P E G polyethylene glycol PFG pulse field gel electrophoresis pfu plaque forming units P H potential of hydrogen Pi isoelectric point PKS polyketide synthase PMSF phenylmethylsulfonylflouride pyrG orotidine 5'-phosphate carboxylase encoding gene RACE randomly amplified cDNA ends RAPD randomly amplified polymorphic D N A rDNA ribosomal deoxyribonucleic acid RFLP restriction fragment length polymorphism RNA ribonucleic acid rpm revolution per minute RT reverse transcription - xvi -StoT s serine SAP secreted aspartyl proteases SD scytalone reductase SDS sodiumdodecylsulphate SMA skim milk agar sp species (singular) spp species (plural) T thymine T tyrosine Taa Taq D N A polymerase TCA tricarboxylic acid T-DNA transfer D N A T E Tris-EDTA buffer Ti Tumour inducing plasmid Tris ttis-(hyckoxymemyl)-aminoethane U t o Z u unit URA uracil URA3 Orotidine-5'-Phosphate decarboxylase X any amino acid - XVII -Acknowledgements I am grateful to all of the support and assistance provided to me by my supervisor, Dr. Colette Breuil. Dr. Breuil was always willing to help, to make time for me and provided much needed constructive criticism of this thesis and my research papers. I am grateful to my committee members, Jim Kronstad and Steve Withers, for finding the time to help me through this work. I am also very grateful towards several members of our group, including Peter Loppnau with whom I exchanged many ideas, Phillipe Tanguay who assisted me with several aspects of my work, Carl Fleet who was a great friend and was always willing to provide company when I needed a break, and Seong Hwan Kim. I was assisted in this thesis by several students including Nancy So, Jennifer Evans and Zachhary Hickman and would like to thank them for their help. I would like to thank my parents without whose support I don't know if I would have made it. Also, I would like to very much thank my best friend and greatest motivator, my wife Jennifer. This work was funded by a Natural Science and Engineering Research Council of Canada strategic grant. Personal financial support was funded by a B.C. Science Council Graduate Research Engineering and Technology award with industrial support from Forintek Canada Corporation. Chapter 1 : General Introduction and Research Objectives 1.1. The Problem of Saps tain to the Forest Products Industry The forest products industry is one of Canada's largest industries, generating 62.8 billion dollars and exporting over 40 billion dollars worth of forest products every year [1]. In Canada, over 250, 000 people are direcdy employed by the forest industry [1]. Approximately 30% of our exports are softwood lumber, placing Canada as the largest exporter of softwood lumber in the world. To maintain its markets, the Canadian forest products industry must be able to provide a high-grade defect-free product; however, wood is often colonized by sapstaining fungi which produce a dark blue to black discoloration that decreases the value of the wood (Figure 1-1). Also, these fungi raise concerns in countries importing wood products, as they can be non-indigenous and could cause an epidemic. The Canadian forest products industry spends millions of dollars annually on fungicides to control the stain caused by these fungi. Currentiy, six fungicides are registered for sapstain prevention by Agriculture Canada. These include: didecyldimethylammonium chloride, borax or borate, azaconazole, 3-iodo-2-propynylbutylcarbamate, sodium carbonate Figure 1-1: Example of sapstain in Pinus contorta A) a 2x4 and B) a log in cross section. and 2-(tMocyanomemylthio) benzothiazole. These chemicals do not have the duration of effectiveness desired by the industry and still present significant environmental concerns. Kiln drying is the most common alternative to the use of antisapstain chemicals. This process lowers the moisture content of the wood to below 20% at which point contaminating fungi are unable to grow. Rewetting of the wood during transportation or storage can occur and it is not uncommon for shipments of kiln-dried wood to become stained. Kiln drying is also an expensive process and is only feasible for wood species for which the profits are high enough to overcome the associated costs [2]. Given this, several alternative treatments are being examined, including the use of biological control agents, such as albino fungi [3-5]. So far these treatments have had limited success as these agents can be slow growing and beyond the laboratory have been shown to be unreliable [6-8]. More information about the relationship between the biological control agent, mvading organisms, the wood substrate and the environmental conditions at the site of application are needed to improve these results [9]. With proper implementation and optimization biological control agents might, in future, be effective in protecting freshly cut trees, especially at the harvesting site where chemicals cannot be used. More recently, the implementation of integrated pest management strategies has been promoted. These strategies include avoiding the storage and harvesting of logs during high-risk periods, the rapidly handling of logs in warmer climates and water saturating logs by ponding. These control strategies are used in conjunction with chemicals and other treatments when necessary. It is likely that the best method for controlling sapstain will integrate these current strategies with new methods developed from an increased understanding of the physiology of sapstaining fungi. 1.2. Sapstaining fungi 1.2.1.Classification of Staining Fungi Over 200 fungal species have been associated with sapstain and have been subdivided into three major groups: (1) Species of Ceratocystis, Ceratocystiopsis, and Ophiostoma (2) Black yeasts including Aureobasidiumpullulans and Phialophora spp. (3) Dark moulds mcluding Alternaria alternata and species of Cladosporium [10] [2]. The Ascomycetes Ophiostomatoids and their anamorphs are able to penetrate deeply into sapwood and are commonly isolated throughout Canada. This group is a major problem to the forest products industry and consists of Ophiostoma spp., Ceratocystis spp., and the anamorph Leptograpbium spp. In a 1997 countrywide survey done by our group and our collaborators Ophiostomapiceae was the most frequently isolated species on logs and lumber, while Ceratocystis coerulescens, and Leptographium spp. were more often associated with deep stain in freshly cut logs [11]. Ceratocystis and Ophiostoma have been separated as two distinct genera based on morphological, physiological and genetic evidence. An easy method to differentiate these genera is through their growth on cyclohexirnide, as Ophiostoma species are resistant to this antibiotic whereas Ceratocystis species are not [12], As well, the cell walls of Ophiostoma spp. contain rhamnose and cellulose whereas in Ceratocystis spp. these carbohydrates are absent [13]. Phylogenetic analyses based on the rDNA sequences of these species supports the distinction of these genera [14-16]. A PCR-RFLP based method separating the commonly isolated genera of sapstaining fungi Aureobasidium, Ceratocystis, Leptographium and Ophiostoma has been developed [17]. It is often difficult to differentiate many species of sapstaining fungi using morphological characteristics, and this process can be time consuming and requires expertise. Molecular techniques have, therefore, been developed to aid in the identification of several sapstaining fungi [17-24]. For example, although morphological and mating tests identified two groups of inter-sterile mating groups in 0. piceae, one isolated predominately from softwoods and the other from hardwoods, the identity of the species in these groups was not clear [25]. Molecular methods such as RAPD, RFLP and phylogenetic analyses were subsequently used to confirm this segregation and identify the members in each group [18-21]. A PCR based method for separating the two groups direcdy from asexual spores collected from wood was subsequently developed [26] [27]. Phylogenetic analysis has also been used to study the relationship of Ophiostoma piliferum with other staining fungi and a PCR-RFLP based method was developed to identify 0. piliferum isolates from wood [17] [22]. Recently, a novel approach using pulse-field gel electrophoresis has been investigated as a method of separating different members of the Ophiostoma genus based on their karyotypes; however, it was concluded that electrophoretic karyotypes do not have taxonomic usefulness [28]. The genetic diversity of 0. piceae and 0. piliferum has also been assessed as a means of discrirrrmaring between isolates with different origins [29] [30]. - 6 -The proper taxonomical position of Leptographium sp. has been debated and phylogenetic analyses have been used to determine that anamorphic Ljptographium species were anamorphs of Ophiostoma species [16]. Further work has confirmed this link and provided a means to identify many Ljeptographium species [31]. Controversy has also existed about the identification of members of the C. coerulescens complex, a group of morphologically similar Ceratocystis species [32]. Phylogenetic analyses and isozyme data were used to show that species of Ceratocystis from the C. coerulescens complex isolated from gymnosperms formed a single clade [32] [33]. Recently PCR-RFLP based methods for identifying species of Ceratocystis have been developed [23] [24]. 1.2.2.J3iology of Sapstaining Fungi In nature species of sapstaining fungi are dispersed in a variety of manners at both tree harvest sites and sawmills. The major vectors for infection of wood tissue are arthropods such as bark beedes, mites and wood borers as well as nematodes. Some sapstain fungi, such as Ophiostoma minus, Ophiostoma ips and Ophiostoma clavigerum have symbiotic relationships with certain bark beetles [34-36]. Water dispersal of spores, contarnination by harvesting equipment and manual post-harvest contamination may also allow the spread of these fungi [37] [34] [2] [38] [9]. Ophiostoma spp. grow and sporulate best on media with a high carbon/nitrogen ratio, although some species only produce perithecia on certain carbon sources such as glucose or lactose [39]. Sapstaining fungi grow best at pH's between 3.5 and 6.5 and at temperatures between 15 and 30 C. Most cannot grow at temperatures above 40 to 50 C or below 4 to 6 C [39] [40]. Sapstaining fungi grow optimally at moisture contents between 60 and 80 percent (based on an oven-dry method). They can, however, survive at moisture contents between 20 and 160 percent, although growth is abridged at moisture contents above 120 %, at which point concentrations of free oxygen are no longer sufficient to support growth [41]. Many fungi, including most species of Ophiostoma, show some form of pleomorphism and are able to grow in a variety of forms. The two most common morphologies for fungal growth are yeast-like growth with cellular division occurring as a budding process, and hyphal growth with cellular division and growth occurring at hyphal apices. A number of factors can mitigate the conversion between the two morphologies, including oxygen supply, pH, and temperature. In nature, and particularly in wood, hyphal growth is the dominant morphology. Hyphal growth involves a substantial number of factors, most of which are not well understood. Turgor pressure is thought to provide the major driving force for the extension of the hyphal tip; however, reports on Saprolegnia ferax have shown that hyphal extension occurs even in the absence of turgor pressure [42]. Concomitant with the extension of the hyphae is the recruitment of vesicles to the hyphal tip, each carrying the enzymes and building blocks necessary for the creation of the new cell wall, while the incorporation of the vesicles allows the extension of the cell membrane. During this process, enzymes critical for the acquisition of exogenous nutrients, are also released from the hyphal tip by the recruited vesicles, which are able to pass through the incompletely cross-linked cell wall. Assimilation of degraded nutritive polymers occurs behind the growing edge of the hyphae as nutrients diffuse across the cell wall or are actively transported into the cell. During hyphal growth, sapstaining fungi often produce melanin in their cell wall, causing the stain observed in wood. Eagen et. al. reported that melanization of Ophiostoma floccosum is dependent on the carbon and nitrogen sources available to the fungi [43]. Inhibitor studies indicated that sapstaining fungi use the 1,8-dihydroxynapthalene (DHN) pathway to produce melanin [44]. Genes encoding enzymes from this pathway, including two reductases, a 1,3,8-trihydroxynapthalene reductase (3HNR), a 1,3,6,8-tetrahydroxynapthalene reductase (4HNR) and a scytalone dehydratase (SD) gene have been cloned and sequenced from 0. floccosum 387N [45-47]. As well, a polyketide synthetase (PKS), a 4HNR and a SD gene have been cloned and sequenced from Ceratocystis resinifera [48]. The PKS gene was disrupted in this fungus, producing an albino phenotype, indicating that the D H N melanin pathway is the only pathway used by C. resinifera to produce melanin. Partial sequence data have also been obtained from the PKS, 4HNR, 3HNR, and SD genes from several other sapstaining fungi [49]. Fungi are able to grow across nutrient poor areas and to produce aerial structures that have no contact with the substrate. Both of these processes occur due to nutrient and water translocation mechanisms in the fungal culture. Extensive work in Hypholoma fasciculare has shown that bi-directional translocation of nutrients occurs to equalize nutrient concentrations between nutrient rich and nutrient poor areas [50]. As staining fungi grow on wood they continually expand searching for new nutrient sources, leaving behind nutrient depleted areas. Similar mechanisms, to those found in H. fasciculare, are likely used - 9 -by sapstaining fungi to provide nutrients for mycelia in nutrient poor areas, and for hyphae searching for new nutrient sources. 1.2.3.Growth of Sapstaining Fungi in Wood Once fungal spores infect wood they germinate producing hyphae that spread through the ray cells, tracheids and other natural passageways in the wood, however, growth preferentially occurs in ray cells due to their high nutrient content [51]. Movement between cells is often accomplished through bordered pits via penetration of the torus of the pit membrane. Direct penetration of cell walls may occur by the formation of appressoria that generate large amounts of turgor pressure that drives a "bore hyphae" or penetration peg through the cell wall. Some evidence of enzymatic involvement has been noted in this process but this has not been substantiated [52]. Sapstaining species are not able to degrade the structural components in wood, such as Hgnin or cellulose, and they have no or incomplete cellulolytic and/or lignolytic degradation pathways [53] [54] [41]. Instead sapstain fungi utilise non-structural wood components and extractives, mcluding triglycerides, fatty acids, proteins, waxes and alcohols [52] [2]. These compounds are mainly found in living or recently dead sapwood parenchyma and can comprise less than ten percent of the dry weight of wood [51]. Triglycerides are the major lipid component in fresh sapwood and comprise over 50% of total extractives in some species [55]. They are an important form of carbon storage in trees - 10-and contain a variety of fatty acid moieties and over twenty different fatty acids have been identified in wood [56]. To utilise these wood components, sapstaining fungi produce Upases and esterases, which break the triglycerides down into their component fatty acids and glycerol. A strain of O. floccosum 387N has been shown to produce an extra-cellular lipase able to degrade wood triglycerides [57] [58]. As well, O. floccosum reduced triglycerides and fatty acids by 75 and 60 percent, respectively, after a two-week incubation on Pinus contorta sapwood [59]. Cartapip™, an albino strain of O. piliferum, reduced triglycerides and fatty acids by 75 to 100 percent after incubation on Southern Yellow pine {Pinus taeda and Pinus virginiand) wood chips for two weeks [60] [61]. Steryl esters and waxes comprise a very low percentage of sapwood (~0.5% oven dry weight of wood). Esterases, oxidases and other enzymes have been implicated in the degradation of these compounds [62]. Levels of resin acids in sapwood are also extremely low, but are substantially higher in heartwood, preventing fungal growth [59]. Soluble carbon in trees is often stored as either starch or free sugars and the relative levels of these compounds change with the seasons [63] [64]. Several species of sapstaining fungi have been reported to efficiently degrade xylose, mannose and glucose in P. contorta wood billets [49]. Nitrogen levels in trees are also extremely low, at only 0.01-0.3% of the wood's dry weight, and levels of easily assimilated nitrogen sources, like ammonia, are insufficient to support fungal growth [39] [65]. Trees are known to store most of their nitrogen in an organic form and the majority of nitrogen in trees is found as proteins and arnino acids [66] [67]. These proteins are typically stored as protein conglomerations called protein bodies within parenchyma cell vacuoles [67] [68]. The major protein stored in these bodies is ~32-kDa, indicating that this protein acts as a special storage protein. The total level of this protein and the number of protein bodies has been shown to fluctuate with the seasons, dropping dramatically with spring budding [69] [67]. Proteins act as a source of both nitrogen and carbon for fungi. As fungi are unable to directly assimilate proteins, they excrete a wide range of proteases and peptidases in order to break down exogenous protein sources. These enzymes degrade proteins into short polypeptides and amino acids. Fungi are then able to uptake peptides of six or fewer amino acids in length, and individual amino acids, via specific permeases or transferases [71] [72]. O.floccosum 387N was shown to produce an extracellular protease capable of breaking down wood proteins [70] [58]. Most of the acquired amino acids are used directly in the synthesis of new proteins; however, they may also be used as an energy source through oxidative degradation, especially if there is an excess of amino acids available. In this process deamination of the amino acid produces ammonia and its respective oc-keto acid. This ammonia can feed directly into amino acid synthesis pathways by combining with a-ketoglutaric acid to produce glutamate, which is then converted to glutamine via glutamine synthetase (Figure 1 -2). These reactions, producing glutamate and glutamine, are involved in the utilisation of all Figure 1-2: Reactions involved in the utilisation of ammonia acquired from amino acids. Adapted from Magasanik [73]. - 13 -non-preferred nitrogen sources [73]. The mechanism by which the a-keto acid produced is oxidatively degraded depends on the amino acid. Of the twenty amino acids five different catabolic products are formed: acetyl-CoA, a-ketoglutarate, succinyl-CoA, fumarate and oxaloacetate. 1.3. Regulation of Nitrogen and Carbon Catabolic Pathways 1.3.1.Global Nitrogen Repression and Activation Considering their key roles in amino acid synthesis and nitrogen acquisition, it is not surprising that ammonia, glutamine, glutamate and in some cases asparagine are preferred nitrogen sources for fungi. Glutamate and glutamine are often kept at highly elevated levels within the cell and glutamine plays a key role in nitrogen catabolic repression. Ammonia can also exert strong nitrogen repression but only indirecdy, through the synthesis of glutamine. Glutamine synthesis by glutamine synthetase is under extremely tight regulation and is one of the most complex enzyme regulation systems known. It is not known whether glutamine is able to direcdy cause repression or if it acts through a metabolite. The localisation of the cellular glutamine pool and the identity of the molecules able to sense repressing levels of glutamine are also not known. It is well established that nitrogen repression and derepression act through global-acting GATA-binding transcriptional activators like A R E A in Aspergillus nidulans, NIT2 in Neurospora crassa, as well as Gln3p and Nil lp in Saccharomyces cerevisiae [74] [73]. As well, in S. - 14-cerevisiae the global-acting transcriptional repressors Dal80p and Nil2p act to repress the action of Gln3p and Nil lp [73]. Most fungi are thought to possess homologous GATA-binding factors, and other related factors, to control the regulation of nitrogen catabolic genes. Al l of these transcription factors contain a highly conserved zinc finger domain, common to many transcriptional activators, that allows them to bind to GATA sequences in the promoters of the genes they regulate. Differential regulation of nitrogen catabolic genes is achieved by the organisation and sequence of GATA-containing transcriptional recognition elements. The regulation of GATA-binding factors themselves is either constitutive or autogeneous with their promoters containing GATA recognition elements [74] [74]. Regulation is also achieved by transcript turnover rates, as the half-life of transcripts is ~7 min in repressed conditions and ~40 min in derepressed conditions [74]. Glutamine represses GATA-binding factors either directly or indirecdy. In S. cerevisiae glutamine allows TOR kinase phosphorylated Gln3p to complex with Ure2p. This protects Gln3p from dephosphorylation and localises it to the cytosol [74]. In low levels of intracellular glutamine a conformational change in the Ure2p-Gln3p complex causes its disassociation. This allows the dephosphorylation and subsequent relocalisation of Gln3p to the nucleus, where it is able to affect gene activation [74]. It is thought that Ni l lp is similarly regulated by intracellular glutamate levels [74]. In A. nidulans it was reported that the C-terminal 12 a.a. of GATA-binding factors are critical for repression. As well, key amino acid substitutions in an alpha helical region of the zinc finger caused insensitivity to glutamine repression [74]. - 15-Generally, expression of nitrogen-catabolically repressed genes requires activation by global-acting nitrogen transcriptional activators through the removal of nitrogen repression by the mechanisms listed above. For some genes this is sufficient to allow transcription; however, most systems also require a second inducing signal that allows the selective expression of specific catabolic enzymes. It was suggested that this induction occurs via inducers that bind to pathway-specific regulatory proteins causing their activation. Several pathway-specific regulatory proteins are known. For example, in A. nidulans FACB, NIRA, PRNA, and U A Y are involved in acetate, nitrate, proline and purine catabolism respectively; in S. cerevisiae ARG80, ARG81 and PUT3 are involved agiriine and proline catabolism and in N. crassa NIT4 is involved in nitrate catabolism [73-79]. Al l of these proteins are members of the GAL4 family, contain a Cys/Zinc binuclear zinc cluster, have a C-terminal activation domain and an N-terminal D N A binding domain and may interact with other global-acting elements to cause the expression of specific catabolic genes. These regulators are generally constitutively expressed but are only activated upon the presence of specific inducers. 1.3.2.Regulation of Carbon Catabolic Pathways Like many other microorganisms fungi tighdy control the expression of genes involved in carbon catabolism. In the presence of favoured carbon sources global-acting repressor proteins, such as CREA from A. nidulans and the Migl complexes of S. cerevisiae and N. crassa, act as transcriptional repressors of genes involved in catabolism of non-favoured carbon sources [80-82]. This phenomenon is often called "glucose repression" as glucose is a highly favoured carbon source and is highly repressing to other carbon catabolic genes. - 1 6 -CREA from ^4. nidulansis 415 a.a. in length with two zinc finger motifs, as was reported for Migl from S. cerevisiae [80]. CREA has a consensus binding site of 5'-SYGGRG-3'; however, it may also bind to similar non-consensus sequences. It is likely that other proteins are involved in carbon repression in A. nidulans, such as CREB and CREC and CCAAT binding proteins and work needs to be done in order to confirm the roles of these proteins. In S. cerevisiae and N. crassa carbon repression is affected by the Migl complex [81]. In S. cerevisiae this complex is formed of Migl , Tupl and Cyc8. The formation of this complex is pardy regulated by the localisation of Migl , which is determined by its phosphorylation state. In repressing conditions Migl is unphosphorylated and localised in the nucleus where it can complex with Tupl and Cyc8 and repress carbon catabolic genes. Under non-repressing conditions Migl is phosphorylated by the Snfl complex, and is subsequently shuttled to the cytosol where it is unable to cause gene repression. In N. crassa the Migl complex is formed by Migl , Tupl, and Ssn6 [82]. The formation of this complex is regulated in a similar manner as in S. cerevisiae, with Snfl phosphorylating Migl and causing its relocalisation. 1.4. Fungal proteases 1.4.1.Proteases Proteases are widely produced amongst fungi and serve a number of roles, including nutrient cycling, activation of zymogens, post-translational processing and cell autolysis [83] - 17-[84-86]. There are four major types of proteases: aspartyl or acidic proteases, cysteine proteases, serine or alkaline proteases, and metallo-proteases. Some of these groups are very diverse with members that have a wide range of substrate specificities, as well as, pH and temperature optima. Other groups tend to be small and conserved. Serine proteases are described in more detail in the following section. 1.4.2. Serine Proteases Serine or alkaline proteases have an essential serine residue at their active site within a catalytic triad that includes a lustidine and an aspartate residue (Figure 1-3). Serine proteases are the most common type of protease and are produced by most classes of organisms, including viruses, bacteria, mammals, and plants. Most of these proteases are between 18 and 35 kDa and are typically most active at neutral to alkaline pH's with pi's between 4.4 and 6.2. Most serine proteases tend to be translated as zymogens and require proteolytic activation. Glycosylation of serine proteases is not common. Al l serine proteases are strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropylphosphorofluoridate (DFP). Figure 1-3: A schematic representation of catalytic triad of a serine protease, showing the hydrogen bonding that occurs between the members of the triad [87]. - 1 9 -There are five clans of serine proteases, with the two major clans being the trypsin and the subtirisin clans. These two clans differ in the order of the catalytic triad residues in the protein and in the 3D scaffolding that holds these residues. The exact orientation of the catalytic residues is critical for the functionality of all serine proteases. In trypsinases the catalytic triad is ordered as Ser-His-Asp in the linear sequence of the protein and these residues are held within the scaffolding between two (3 sheets. In contrast for subtilisins, or subtilases, the catalytic triad is present in the exact opposite order as Asp-His-Ser, which are held within a a-helix and [3-sheet scaffold (Figure 1-4) [88]. Trypsinases and subtilases also vary considerably in their substrate specificity, as trypsinases tend to be highly specific, while subtilases tend to have a broad substrate specificity. The catalytic mechanism of serine proteases is well understood (Figure 1-5) [87]. In short, the catalytic histidine residue acts as a proton acceptor for the active site serine, allowing it to perform a nucleophilic attack on the carbonyl carbon of the scissile bond. This results in the formation of a tetrahedral intermediate, which is stabilised by the positive charge on the histidine residue. This intermediate then collapses into an acyl-enzyme intermediate through acid catalysis and proton donation from the histidine residue, releasing the first portion of the substrate. Deacylation then occurs with the nucleophilic attack of water, which is activated by deprotonation by the histidine residue. This second nucleophilic attack produces another tetrahedral intermediate, which then breaks down to release the second portion of the now cleaved substrate. - 2 0 -Figure 1-4: A schematic representation of the 3D structure of a generic subtilase, showing a-helices as cylinders, and [3-sheets as arrows. Also shown are the relative locations of the catalytic triad and substrate binding sites in the protein sequence [91]. - 2 1 -tap / H i s OH I in / H i s C H , >W Sor S H Q / . Asp . . 0^2/ H^vf ' \®-»/ 6' Tetrahedral Intermediate Asp \ = t J : C H , C , 0* O, b-H £ 4 First Product Leaves Asp / H i s C H , \ = N : 0 VV°" Second Product Leaves Sty O Asp / H i s ! H , C H . / 54 0 Tetrahedral Intermediate Asp Vioj/ / H i s C H , V = N : Sor C H , r Figure 1-5: A schematic representation of the catalytic mechanism of a serine protease cleaving the scissile bond of a peptide. Adapted from Warshel [87]. - 2 2 -1.4.3.Subtilases Subtilases are a family of serine proteases named after the enzyme subtiksin from Bacillus subtilis. Currendy over 200 subtilase sequences are known [88-90]. Based on sequence alignments Siezen and Leunissen suggested the division of the subtilase clan into six subfamilies: (1) the subtilisin family that contains enzymes from Bacillus, (2) the thermitase family found only in micro-organisms, (3) the protease K family of mainly fungal endopeptidases, (4) the lantibiotic peptidase family specialized in cleaving leader peptides from precursors of lantibiotics, (5) the kexin family which is a large group of proprotein convertases and (6) the pyrolysin family which is a varied group with low sequence conservation [89]. There are several highly conserved residues present in most subtilases, but only the catalytic triad and a glycine residue near the catalytic serine are totally conserved. Four other glycine residues are substituted only once or twice, and several other residues are substituted minimally and usually only with structurally similar residues. 1.4.4.Fungal Subtilases Subtilases in fungi typically belong to the protease K family. The most widely known member of this family is protease K produced by Tritirachium album [92]. This family shares an overall protein sequence similarity of over 37%; however, conservation around the active -23 -site residues is very high. There are a total of 41 invariant residues between members of the family. Regions of low homology tend to occur on the protein surface, in loops between structural a-helixes and p-strands. A few members of this family have C-terminal extensions beyond their catalytic domain. These extensions likely play a role in the vacuolar targeting of the enzyme, as was reported for PRTB from S. cerevisiae and PEPC from Aspergillus niger [93]. Al l the fungal subtilases identified so far are translated initially as pre-pro-enzymes. Pre regions are short, composed of 15-20 a.a and generally contain a hydrophobic core of nine amino acids. Usually, they have a predicted (3-sheet structure preceded by positively charged amino acids. A signal peptide cleavage site (Leu-X-Ala) occurs after the termination of the (3-sheet structure. Pro regions are typically around 100 a.a. in length and play a dual role. First, pro regions have been shown to be critical in the proper folding of mature subtilases [94] [95]. Although unstructured on their own, Pro regions form four-stranded antiparallel (3-sheet structures with two three turn a-helices when complexed with subtilases, stabilising the <x[3a core of the subtilase [96]. The second role of the Pro region is to inactivate the enzyme by blocking its active site until it is released from the cell and the pro region removed [96]. Removal of signal sequences may be autocatalytic or involve another peptidase enzyme [97]. Protease K is commercially important due to its stability and ability to digest even the most recalcitrant substrates. The crystal structure of this enzyme has been determined and revealed that protease K has an overall shape of a half sphere with no discernible domains -24 -Figure 1-6: A ribbon schematic representation of the 3D structure of protease K , showing the locations of the catalytic triad. The amino acid sequence of protease K was obtained from NCBI and Rasmol was used to draw the structure of protease K. - 2 5 -(Figure 1-6) [91]. Eight parallel (3-sheets form the core of the enzyme and are surrounded by five a-helices and three and parallel |3-sheets. Al l the catalytically relevant residues were found at the terrrrini of secondary structural elements. The geometry of the catalytic triad, Asp39-His69-Ser224, was essentially identical to that of the prokaryotic enzyme subtilisin and of the mammalian enzyme chymotrypsin [98]. The substrate recognition site was defined by two peptide sequences, GlylOO-SerlOl and Serl32-Glyl34, which link the central ^-sheets with peripheral a-helices. These two peptide sequences are in a parallel orientation allowing the substrate to easily form the necessary hydrogen bonds by binding in an antiparallel fashion. The S, site is formed by a large loop from Asnl61-Prol71, which gives protease K its non-specific nature, common in subtilases. The S2 site is formed as a shallow groove and no S3 site is formed as P 3 residues would point away from the recognition site. Protease K is known to have four different calcium co-ordination sites and calcium is essential for the activity and stability of the enzyme. Two disulfide bonds that form between residues 27-118 and 175-247 are also critical to the stability of protease K. The fungus T. album has been shown to produce two other subtilases aside from protease K, protease R and T. The structure of protease T is similar to protease K , but protease R contains considerably more a-helices [92]. Subtilases have been characterised from a number oi Aspergillus species due to their commercial and medical importance. A. niger a commonly used host for the industrial scale heterologous expression of proteins has been shown to contain at least two subtilase genes, PEPCand PEPD [99] [100]. PEPCis constimtively expressed, localised in the vacuole and plays a housekeeping role [99]. This gene has a significant C-terminal extension similar to -26-PRTB from S. cerevisiae. PEPD shares only 55% nucleotide similarity with PEPC, but shares a high degree of identity with subtilases involved in pathogenicity [100]. A., fumigatus isolates from patients with aspergillosis produce subtilisin like proteases that play a role in the infection process. The alkaline protease gene AFAlp from A. fumigatus was cloned and disrupted. The obtained disruptants were deficient in extracellular proteolytic activity over a wide range of pH's and were unable to produce the encoded 33 kDa alkaline protease [101]. The pathogenicity of the disruptants though was no different from the parent isolate, using a murine model. The authors suggested that the ability of the alkaline protease deficient strain to grow in lung tissue might be due to the microenvironment in the lungs stimulating the production of acid or extremely alkaline proteases, thereby compensating for the disruption of the alkaline protease gene [102]. Other researchers also identified an extracellular serine protease produced by A. fumigatus, and indicated that this was likely the only extracellular serine protease produced by the isolate used [103]. The authors disrupted this gene using chemical mutagenesis and reported a significant loss in the virulence of the strain [103]. The different results obtained from these two studies may reflect the random nature of chemical mutagenesis, which could have mutated important regulatory or secretory elements, instead of targeting an alkaline protease gene as was done in the first study. Two other subtilase genes have also been identified in alternative strains of A. fumigatus; including ALP and ALJP2 that was shown to be involved in fungal development [104] [105]. At least three different extracellular alkaline proteases are produced by A. nidulans and the gene prtA that encodes a serine protease has been cloned from this fungus [106]. Protease - 2 7 -activity is critical for the proper production of the flavours of soy sauce, and the A. ory^ae alkaline protease A L P is critical in the process. The Alp gene was cloned and heterologously expressed in S. cerevisiae [107]. A serine protease gene, pepB, has also been cloned from Aspergillus awamori. The disruption of this gene was accomplished using a double homologous crossover strategy with the use of a counter selectable marker. Two disruptants were obtained and both showed significandy reduced degradation of heterologously expressed thaumatin [108]. Subtilases have also been studied as possible virulence factors in several fungal species. For example, a possible pro-hormone processing subtilase-like multigene family, PRT1, has been characterised from Pneumocystis carinii, a medically important fungus that causes pneumonia in immunocompromised individuals [109]. Copies of the genes in this family varied slightly between isolates and were distributed throughout the genome. Pyrenope^i^a brassicae, the causative agent of light leaf spot disease in brassicas contains an intracellular subtilase gene similar to PEPC, Psp2, which is involved in vegetative growth and sexual development [93]. Another gene similar to PEPC was cloned from Podospora anserina using a PEPC probe and is involved in vegetative incompatibility [110]. Magnaporthepoae, a root pathogen of Kentucky bluegrass produces a subtilase, M p l , during infection [111]. Two very similar subtilases have been identified in the nematophagous fungus Veriicillium chlamydosporium and the entamophagous fungus Metarhitnum anisopliae [112]. Different strains of V. chlamydosporium produce very different levels of extracellular serine protease activity, and between two and four subtilase genes were identified in these strains [90]. In M. anisopliae three isoforms, Pr1A, Pr1B, and Pr1C, of a subtilase gene have been reported [113]. Pr1A was shown to be - 2 8 -polymorphic with variations between geographically distant isolates, while Pr1B and Pr1C varied only slighdy between geographically distant isolates. A subtilase gene,prb1, related to mycoparasitism has also been cloned from T. haryianum [114]. As well, subtilase genes have been cloned from the Crayfish plague fungus Apbanomyces astaci, the commercially important edible mushroom Agaricus bisporus, the antibiotic cephalosporin producing Acremonium cbrysogenum, and the wood decay fungus Schi^ophyllum commune [115-118]. A subtilase produced by the sapstaining fungus 0. floccosum 387N was purified by FPLC using hydrophobic interaction chromatography [70]. The enzyme was 33 kDa with a pi of 5.6 and was inhibited by common serine active site inhibitors such as PMSF, while the addition of calcium increased its reaction rate [91]. The enzymes substrate specificity was similar to protease Ks with apolar and aromatic residues preferred at the PI position. This enzyme was able to autocatalytically cleave itself into two major fragments. The N-terminal sequences of both fragments were obtained and had high homology with other protease K subfamily subtilases. Polyclonal antibodies were raised against this protease and used to localise the enzyme to the cell wall and extra-cellular sheath of the fungus when grown on four different softwood species [119] [70] [120]. 1.4.5.Regulation of Fungal Protease Genes Protease genes are typically regulated by both induction and derepression [83]. In particular the expression of proteases can be regulated through carbon, nitrogen and sulphur - 2 9 -repression and induction by specific protein sources, especially host tissues [121-123]. Pathway-specific signal molecules involved in the induction of protease genes by exogenous protein sources are unknown at this time; however, peptides derived from the protein source have been reported to provide the inductive signal [74]. This inducing signal can be sufficient for the expression of some protease genes. Environmental factors such as pH and temperature can have an overriding effect preventing gene expression [124] [125]. Proteases that play a role in cell autolysis are expressed only in dead or dying mycelia, while protease genes involved in intracellular protein turnover, signal processing, and other housekeeping roles are generally constitutively expressed [118] [126]. The regulation of the secreted aspartic protease (SAP) gene family in Rhi^opus oty^ae has been well characterised [121]. This family of proteases are critical for the production of quality tempe from soy beans and consists of four members (SAP 1-4) which share 76-92 % amino acid identity [121]. Specific probes were designed for each gene and used in Northern analyses to determine the regulation of each of the four genes. pH was an overriding determinant in the expression of sap1-3, and these genes were not expressed at pH's higher than 4.5, even in the presence of inducing exogenous protein. Inorganic sources of nitrogen and sulphur also strongly repressed sap1-3. Exogenous protein induced sap1-3, but only in the absence of nitrogen or sulphur repression. Extracellular protease activity did not coincide with increases in sap gene transcription, suggesting that the translation or degradation of the encoded proteins may be closely controlled by alternative mechanisms. The expression of sap4 occurred at low levels in all the conditions tested but decreased under conditions of nitrogen or sulphur derepression indicating that this gene is regulated separately from sap1-3 [121]. - 3 0 -C. albicans also has a well-characterised SAP gene family. C. albicans causes infections of several mucosal surfaces, including respiratory, oral and vaginal tissues causing debilitating and recurrent diseases [127-130]. The SAP gene family in this fungus consists often members (sapl-10) and SAP2 is the major transcript [130]. Expression of sap2 was induced by exogenous protein, and studies showed that SAP2 plays a dominant role in the pathogenicity of C. albicans [129]. sapl and sap3 were expressed during phenotype switching from yeast to mycelial growth [130]. During hyphal growth sap4-6 were expressed at neutral pH's even in the absence of an inducing protein source. These genes were not transcribed during yeast-like growth. The expression of sap8 was regulated by temperature, whereas sap9 and sap10 were constitutively expressed at low levels [97] [130]. The regulation of the aspartyl protease, aspA, from Penicillium roqueforti has also been studied [131] [132]. This gene was not repressed by glucose, but was strongly repressed by nitrogen, even in the presence of inducing protein sources. Alkaline pH's also repressed the gene in the presence of inducing protein sources; however, this repression was due to the lack of the production of inducing peptides at alkaline pH and not direcdy due to pH repression. In alkaline conditions the PRO region of ASPA is not cleaved off, preventing the activation of the enzyme. This stops the production of inducing peptides by the mature enzyme, preventing the induction of aspA [132]. Addition of mature ASPA to alkaline cultures of P. roqueforti produced inducing peptides and lead to the expression of aspA. A constitutively expressed aspartyl protease gene, aspS, was identified in Sclerotinia sclerotiorum [133]. The regulation of a non-aspartyl acid protease gene, acpl, from this fungus has also - 3 1 -been studied. This gene was induced by the presence of host tissue, repressed by alkaline pH's and regulated by both carbon and nitrogen repression [134]. Proteases are important in the vegetative incompatibility reaction in P. anserina, and work has been done to determine whether specific proteases are induced during this reaction. An aspartyl protease gene thought to be involved in this process, papA, from P. anserina was reported to be induced upon carbon derepression, regardless of the presence of inducing protein [123]. The induction of this gene during carbon starvation was reduced by suppressers of the vegetative incompatibility reaction, but this was discovered to be through a general mechanism and not specific to papA. A serine protease gene, pspA, was induced during the vegetative incompatibility reaction, and it was suggested that PSPA plays a role in the cell lysis reaction during vegetative incompatibility [110]. Work has also been done on a variety of fungal subtilases. The prtA gene from A. nidulans, which encodes an extracellular subtilase, is regulated by pH; however, even at low pH the expression of this gene can be induced by nitrogen starvation [135] [136]. The subtilase gene, psp2, from P. brassicae was expressed under all of the conditions tested indicating that it may play a housekeeping role [93]. Production of the subtilase PR1 by M. anisopliae is induced by insect cuticle or chrtin, but not by other exogenous protein sources such as BSA or casein, suggesting that this protease plays a role in cuticle degradation [122]. Chitin and cell walls of Rhi^octonia solaniinduce expression ofprbl from T. hartnanum, indicating that this gene may play a role in the mycoparasitism of T. har^ianum [114]. The expression of AaSP2 horn A. astaci was also induced by host tissue, in this case crayfish plasma [115]. This gene was not regulated by either nitrogen or carbon repression. A second subtilase gene, AaSP1', - 3 2 -from this fungus was also studied and was expressed at low levels in all conditions tested [115]. A subtilase gene, spr1', from the commercially important fungus A. bisporus was upregulated in the stipe of the sporophore, and was reported to be important in the metabolism of senescing mushrooms [116]. 1.5. Research approach and objectives Nitrogen is vital for the growth of living organisms. As previously mentioned, wood contains very low levels of nitrogen, and sources of inorganic nitrogen are not sufficient to support fungal growth. The ability of sapstaining fungi to utilise organic nitrogen sources is probably critical for their growth in wood. Previous work has shown that sapstaining fungi are able to produce subtilases, a major group of protease, that have activity against wood proteins. The overall objective of this project was to determine the physiological roles of subtilases in sapstaining fungi. Litde work has been done on clarifying the roles of proteases in the physiology of sapstaining fungi and what has been done has only observed the phenotypes of fungi grown on different media types that contain high percentages of specific nitrogen sources. These types of studies, however, do not provide information on the physiological significance of specific enzymes or genes and it is difficult to correlate the results with what may occur in nature. To properly determine the role of specific proteases molecular techniques are required. This thesis, therefore, focuses on the use of molecular techniques to determine the distribution and roles of subtilase genes in sapstaining fungi. -33 -The first objective of this thesis was to gain an understanding of the sequence variability and distribution of subtilase genes in commercially relevant sapstaining fungi. During this work, presented in Chapter 2, three major groups or types of subtilase genes were identified in sapstaining fungi. The second major objective of this project was to determine whether these three groups represented genes with physiologically distinct roles. To achieve this objective three representative genes were chosen, one from each group, and the regulation of each of these genes was determined in response to available nutrient sources, carbon and nitrogen repression, exogenous pH and fungal culture age. These three genes were also heterologously expressed in Escherichia coli in an attempt to determine if the proteins they encode have different substrate specificities or specific activities. The results of these experiments are presented in Chapter 3. Two of the three genes studied were identified as likely being involved in nutrient acquisition from wood. Targeted gene disruption techniques were then used to determine if the proteins encoded by these two genes were important in the growth of sapstaining fungi in wood. In this work the opill gene from 0. piliferum was disrupted, and the characterisation of the mutants generated is shown in Chapter 4. - 3 4 -Chapter 2 : C l o n i n g and genetic analysis of subtilases i n sapstaining fung i 1 2.1. Introduction Subtilase genes have been cloned from several species of fungi. Extensive work has been done on the proteases of Aspergillus species due to their medical and commercial importance. The proteolytic systems of A. nigerhave been studied as this fungus is commonly used in the industrial heterologous expression of proteins, which can be degraded by the proteases produced by this fungus. Two subtilases have been cloned from A. niger, one was reported to be intracellular and the other extracellular [99] [100]. A. fumigatus is, a pathogen of imunocomprimised individuals and different isolates have been shown to contain different subtilase genes. Four subtilases have been cloned horn. A. fumigatus, three are extracellular and have been implicated as possible pathogenicity factors, while the fourth is intracellular and involved in fungal development [101] [103-105], As well, extracellular subtilase genes have been cloned from both A. nidulans and A. ory-^ae [106] [107]. Genes encoding extracellular subtilases have also been characterised from several pathogenic fungi, including P. carinii, P. brassicae, V. chlamydosporium, M. anisopliae, M.poae and several others [109] [93] [111-113]. Most of the enzymes encoded by these genes have been implicated in pathogenicity. Other intracellular subtilases have been cloned from only a few fungi, including P. carinii, P. brassicae and P. anserina, which were reported to play a role in pro-1 Portions of the work presented in this chapter have been published: Hoffman, B., Breuil, C. 2002. The cloning and genetic analysis of subtilases in sapstaining fungi. Curr Genet 41:168-175. Hoffman, B., Breuil, C. 2003. Analysis of the Distribution and Regulation of Three Representative Subtilase Genes in Sapstaining Fungi. Submitted to Fungal Genetics and Biology. Hoffman, B., Breuil, C. 2003. The Roles of the Two Subitilase Genes, opt/1 and opi/2, in Ophiostoma piliferum's Growth on Wood. Submitted to Molecular Plant-Microbe Interactions. - 3 5 -hormone processing, fungal development and vegetative incompatibility, respectively [109] [93] [110]. From these reports it is clear that two general types of fungal subtilases exist; those that are extracellular and perhaps involved in pathogenicity, and those that are intracellular and involved in a variety of housekeeping roles. One author reported the possible segregation of extracellular and intracellular subtilase sequences using phylogenetic analysis but only a few fungal subtilase sequences were used and this required verification [110]. Two major types of PRO regions have also been identified in subtilases but the functional significance of these groups was not known [89]. Although subtilases are common, not all fungi have subtilases as their major extracellular proteases. In C. albicans and R. ory^ae aspartyl proteases are the dominant extracellular proteases [130] [121]. The work presented in this chapter therefore first determines the presence and sequence variation of subtilase genes from 31 isolates of sapstaining fungi. The analysis of 11 partial subtilase gene sequences confirmed the presence of both intracellular and extracellular types. Seven full-length subtilase sequences were then acquired through library screening and RACE-PCR techniques and analysed. The copy number and distribution of homologs of representative genes in other sapstaining fungi was also determined. - 3 6 -2.2. Materials and Methods 2.2A. Fungal and bacterial strains The fungal strain O. floccosum 387N, isolated from softwood chips at the MacLaren Mill (Mason, Quebec, Canada), was obtained from the Forintek Canada Corp. culture collection (Ste Foy, Quebec, Canada). Ophiostoma ulmi and Ophiostoma novo-ulmi, which were both isolated form Elm trees in Ontario, Canada, were obtained from Louis Bernier's culture collection at Laval University (Quebec, Canada). All other fungal isolates were obtained from culture collections at U.B.C. Wood Science Department (Vancouver, Canada). These strains were A. pullulans isolates 72, 123-436, and 156-127; C. resinifera isolates 157-152, 123-22-12, and 125-214; 0. floccosum isolates 55-1 and 197-3; 0. minus isolates 58-4a, 198-4, 123-43-13, and 123-151; 0. piceae isolates 55-3, 157-241, and 123-142, as described in Kim and Breuil [20]; 0. piliferum isolates 55-2b, 80-3, and 156-112; Ophiostoma setosum isolates 160-2a, 55-6-la, 55-6-lb, 160-38a, and 160-38b; Ophiostoma coronatum isolates 68-2, 195-7a, and 195-7b; and Eeptographium spp. isolates 123-239,156-234, 55-5, and 71-15. E. coli TOP10 (Invitrogen) was used for cloning all PCR products. E. coli DH5a (Life Technologies) was employed in all subcloning experiments, and E. coli LE392 (Stratagene) was used in library screening. - 3 7 -2.2.2. Fungal and bacterial growth conditions in culture Fungi were grown at 18-23 C for 2-5 days on malt extract agar (MEA) (Oxoid) plates overlaid with cellophane (BioRad). Fungi were stored at -80 C in 10 % glycerol for long-term storage and in water at 4 C for short-term storage. Bacterial strains were grown on LB agar plates containing 100 ug/ml of ampicillin, except E. coli strain LE392 that was grown without any antibiotic. In liquid culture bacterial strains were grown in 5 ml of LB with 100 Ug /ml of ampicillin. E.coli strain LE392 was grown in LB supplemented with MgS0 4 and maltose. 2.2.3. Purification of Genomic D N A and Total RNA from Fungal Strains Fungal species were grown on M E A cellophane plates for 2-5 days, ~200 mg of mycelia were then aseptically harvested from the surface of the plate, placed in 2 mL cryo micro tubes (Sarstedt) and frozen at -80 C until needed. Genomic D N A was purified from frozen mycelia using the method of Kim et al [17], with a two-stage phenol chloroform extraction prior to D N A precipitation by isopropanol to obtain high purity D N A . Total RNA was purified using a Qiagen Plant RNAeasy extraction kit according to the supplied protocol. Approximately 45 ug of Total RNA was obtained from 200 mg of - 3 8 -mycelia. Total RNAs were periodically checked for quality in 1.2% denaturing formaldehyde gels according to Ausubel et al [137]. 2.2.4. Purification of plasmids from bacterial strains For isolation of less than 10 Ug of plasmid D N A , 5 ml cultures of the appropriate strain were processed using a Qiagen Qiaspin plasmid minikit. When more than 10 pg of plasmid D N A was required 25 ml cultures were processed with a Qiagen Plasmid Miniprep kit. Both procedures were done according to the manufacturers recommendations. 2.2.5. Purification of X phage D N A Approximately, 107 phage were added to 0.5 ml of E. coli LE392 at an O D 6 0 0 of 0.5-0.8. Phage were then allowed to infect the bacterial cells at 37 C for 30 minutes. This mixture was added to 100 ml of LB supplemented with 10 mM MgS0 4 and incubated at 37 C while shaking at 300 rpm. Growth was checked after 4 hrs, if lysis had not yet occurred another 107 phage were added and growth allowed for another three hours. After lysis or after seven hours of growth the cultures were spun at 8,000 X g for 10 minutes to pellet all the bacteria and bacterial particles created during lysis. The supernatant was then collected and X phage D N A was purified using a Clonetech X D N A purification kit. - 3 9 -2.2.6. Polymerase Chain Reactions (PCR) and Randomly Amplified cDNA Ends-Polymerase Chain Reactions (RACE-PCR) The primers used are listed in Table 2-1. PCRs were carried out in a Hybaid TouchDown Thermocycler (InterScience) in 50 uL volumes containing: 40 pmoles of each primer, 200 ng of template D N A , 4 mmoles of dNTPs, 1U of Thermostable Taq D N A polymerase (Rose Scientific) and the supplied I X Thermo D N A polymerase reaction buffer. PCR reactions started with a denaturing step (4 minute at 94 C). Then 35 cycles of denaturing (94 C for 50 seconds), annealing (56-64 C for 50 seconds), and extension (72 C for 50 seconds) were carried out. The PCR reaction ended with a 72 C step for 10 minutes to ensure complete synthesis of all D N A fragments. For RACE PCR reactions, the SMART-RACE cDNA amplification kit (CLONTECH) was used. One ug of total RNA was used to prepare 3' and 5' RACE-Ready cDNAs according to the manufacturer's instructions. For amplification of 5' and 3' RACE-Ready cDNAs, the manufacturers protocol was followed using the PCR reaction conditions: 5 cycles of a 30 seconds at 94 C and 3 minutes at 72 C; 5 cycles of 30 seconds at 94 C, followed by 30 seconds at 70 C and then 3 minutes at 72 C; then 25-30 cycles of 30 seconds at 94 C, followed by 30 seconds at 68 C and then 3 minutes at 72 C; with the final step of 10 minutes at 72 C to ensure complete synthesis of all D N A fragments. Re-amplification of - 4 0 -Table 2-1: List of synthetic oligonucleotides Name Origin Sequence Degenerate P r i m e r s P R O l H o m o l o g y near N - t e r m i n u s A C I C A ( A / G ) A C I G G ( T / C ) C C ( T / C ) T G G P R 0 4 H o m o l o g y at serine active site ( A / T ) G G I G T ( G / A ) G C C A T G G A ( A / G ) G T A C P R Q 7 H o m o l o g y A C G T A C G G T G T G G C C A A G A A Sequencing P r i m e r s P R S 1 ofloc387N A C G T C A T T G C C G G T G T T G A G T P R S 2 ofloc387N C A C G G C A G C G T T A A C A T C A G C P R S 3 of/oc387N A C T C C G A G T A C T C G G T T G C T C P R S 6 ofloc387N A C T C A A C A C C G G C A A T G A C G T P R S 8 ofloc387N A A G G T A C C A A A G T T C A G C T G C Specif ic P r imer s w P 2 - 3 opi/2 C C T T G G G G T C T C G C T C G T A T C T C C w P 2 - 6 opi/2 G T T G G G G G T G T C C T T G G G C A G w l - 1 opi/1 C C C T G G G G C A T C T C G C G C w P l - 4 opi/1 G G T T G C C G T T G A A A G C C A G C T w P i c - 1 opic T C G G G C T C T A C C T G G G G C C T G G G w P i c - 2 opic G C G G A A G G T G A T G G C A G C A G G G C w C r - 1 cr A G C C C A G A G A C G G A G A A G G G T G C C w C r - 1 0 cr G T T G G C G G G C A G G T C G G T G A G Purchased R A C E - P C R pr imers S U P C l o n t e c h C T A A T A C G A C T C A C T A T A G G G C L U P C l o n t e c h C T A A T A C G A C T C A C T A T A G G G C A A G C A G T G G T A A C A A C G C A G A G T N U P C l o n t e c h A A G C A G T G G T A A C A A C G C A G A G T R A C E - P C R pr imers des igned f r o m P R 0 4 / P R 0 7 P C R fragment sequence i n f o r m a t i o n P F 1 ofloc T C G G C A G C G T C G T C G A C A T T C T T P F 2 ofloc A G T G G C C A C G T A G T T G A T G C C A G P C C 1 cr G A C A G C C G T G C C T A C T T C T C C A A P C C 2 cr A G C A A C T C G A C G C C A G C A A T G A P L 1 Ir C A T T G A T G C A G C C G T G C C T A C T T P L 2 Ir A C G C C A G C A A T G A C A T C C G A C A T P P C 1 opic T G T C T G T C G T G G C T T G C T G G C A A P P C 2 opic G A A G A C C T T G A C G G A G A T C A G G T T P P 1 - 1 opill A A C T T T G G C A G C G T C G T C G A C A T P P 1 - 2 opi/1 C A A C G T A G T T G A T G C C G G C A A T G P P 2 - 1 opi/2 T G A C A T C A A G T C C A C C T G G T C G P P 2 - 2 opi/2 G C A C C T T C A T T C A C C A C T G G C A C T G T - 4 1 -generated D N A species was performed using the appropriate gene specific primers and the supplied nested universal primer, using normal PCR reaction conditions, except with an annealing temperature of 68 C. 2.2.7. PCR-RFLP analysis PCR products were generated using the PR07 and PR04 primers at an annealing temperature of 56 C. The resulting bands were excised from the gel. D N A was purified and digested with 1 TJ of Tagl for 2 hours at 65 C. The digested DNA's were then run on 2.5 % agarose gels for 45 minutes at 110 volts. 2.2.8. Vertical Gel Electrophoresis and Purification of D N A from agarose gels Gels were prepared between 0.8 % and 2.5 % agarose depending on the size of D N A species to be separated. Gels were run at 110 volts for between 20 and 45 minutes unless otherwise noted. One pi of Ethidium Bromide was added to minigels and 3 pi to midigels. D N A fragments were visualised under U V light and either photographed with a Polaroid land camera using Polaroid 667 film or with an Ultralum 6000D image analysis system. Appropriate bands were excised from gels under U V with a scalpel. D N A species were then extracted from the gel slice using a Qiagen Gel Extraction kit. - 4 2 -2.2.9. Cloning and sequencing of subtilase gene fragments PCR products were cloned using a TOPO™-TA Cloning® Kit (Invitrogen) according to the supplied protocol. For sequencing, inserts were amplified from the resulting plasmid using the Ml3 forward and reverse primers. The obtained amplicons were then gel purified and 100-300 ng of the extracted D N A was sequenced. Sequencing reactions were carried out with a Perkin Elmer D N A Thermal Cycler 480 using an ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems) at the Nucleic Acid and Protein Service (NAPS) Laboratory (University of British Columbia). Nucleotide sequences were analysed on Applied Biosystem's ABI 373 D N A sequencer (PE Applied Biosystems). Resulting sequences were analysed for homology with other known protein sequences via the BLAST search engine at the National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov). To determine the homology with other known fungal protease sequences, the sequences were then further analysed using P C / G E N E 6.80 (IntelliGenetics Inc) for translation and alignment functions. As well ClustalW, the compute pI/Mw tool (http://expasy.cbr.nrc.ca/tools/pi_tool.html), Genescan, ORFfinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and the predict protein server (http://cubic.bioc.columbia.edu/predictprotein/) were used for further sequence analyses [138] [139]. 2.2.10. Dot blotting and Southern analysis -43 -D N A samples were dot blotted onto Hybond-N+ nucleic acid transfer membranes (Amersham) by the following protocol. Hybond-N+ membranes were first soaked in sterile distilled water before being placed grid-side up on two water soaked 3 M M CHR Whatman filter papers (Whatman) witJiin a dot blot apparatus (Bio-Rad). The membrane was then suction rinsed with distilled water. 0.4 N NaOH was added to each ~2 ug sample of D N A , to a total volume of 200 uL. The samples were heat denatured in a boiling water bath for 5-10 minutes and then placed briefly on ice before being added to the membrane in alternating wells. Samples were alkaline fixed to the membrane by suction filtration. Blank wells contained distilled water. The sample wells were rinsed with 0.4 N NaOH, while the blank wells were rinsed with distilled water. The membrane was then removed from the apparatus, rinsed in 2 X SSC solution for 30 seconds and then allowed to air dry on filter paper for 10-15 minutes. Membranes were then hybridised with the Prot6 probe, as described in section 2.2.15. For southern analyses, 10 pg of fungal genomic D N A was digested with Nco I and Bam HI for O. floccosum (55-1), with Xho I and Bam HI for O. piceae (123-142), with Nco I and Bam HI for O. piliferum (156-112), with Sea I and Bam HI for C. resinifera (125-214), and with Sea I and Bam HI for Leptographium (156-234) and transferred onto Zeta-Probe GT blotting membrane (Bio-Rad) according to the manufacturers recommendations. These enzymes were chosen so that one enzyme would cut the gene at its 3'-end and the other would not cut the gene at all. Digested DNA's were then separated on 0.9% agarose gels at 30 volts for 16 hours, and transferred onto Zeta-Probe GT blotting membrane (Bio-Rad) according to the manufacturers recommendations. - 4 4 -2.2Al. Screening of 0. floccosum 387N X library An 0. floccosum 387N library in EMBL3 (Stratagene) was plated at a density of -2,000 pfu/plate onl50 X 15 mm petri plates [46]. Hybond N+ membranes were used to blot the plates and phage D N A was fixed by oven baking. Membranes were then hybridised to the Prot6 probe, as described in section 2.2.12. After detection, positive plaques were cored and tittered. Secondary plates were prepared at 80-100 pfu/plate on 100X15 mm plates, and screened as described above. The first round of screening was carried out using a hybridisation temperature of 62 C; however, several sets of primary and secondary plates were later screened at 55 C to search for similar sequences. 2.2.12. Hybridisations and Chernnuminescent Signal Detection Dot blots and library membranes were hybridised with a ~400 bp amplicon amplified from 0. floccosum 387N genomic D N A with the primers PRS1 and PR04. The obtained amplicon was labeled with a chemnuminescent probe using the Gene Images Random Prime Labelling Protocol (Amersham) according to the manufacturers' recommendations. The resulting fluorescein-labelled probe was named Prot6. Hybridisation of the labeled Prot6 probe to membranes was performed using the Gene Images random prime-labelling module, version RPN3540PL/95/07 (Amersham) as described in the manufacturers' manual. Hybridisation was allowed to occur overnight (about 16 hours), with rotation, at the appropriate temperature in a T E K Star Hybridisation Oven (Bio/Can Scientific). Stringency washes, blocking, addition of anti-fluorescein-AP conjugate, and Tween-20 washes were performed using a Gene Images™ CDP-Star detection module, version RPN3510PL/98/09 -45 -(Amersham) according to the supplied manual. Washes were done between 50 C and 60 C depending on the stringency required. Signal was generated using a Gene Images™ CDP-Star detection module, version RPN3510PL/98/09 (Amersham) according to the manufacturers recommendations. Exposure to Hyperfilm™ ECL™ high performance chemiluminescence film (Amersham) was used to detect the signals. Southern membranes were hybridised with cr, opill, opil2, and opic probes from amplicons generated using the gene specific primers wCr-l/wCr-10, wPl - l /wPl -4 , wP2-3/wP2-6 and wPic-1 /wPic-2 respectively. Amplicons were gel purified, and then labelled with (32P)a-dATP (3000 Ci/mmole, Amersham) using the Random Primers D N A Labelling System (GIBCOT3RL). Hybridisations with the cr probe were performed overnight at 55 C in 7 % sodium dodecyl sulfate (SDS)/20 mM Na2HP04, pH 7.2. The membrane was washed twice for 30 min each at 65 C in 5% SDS/20 mM Na2HP04, pH 7.2, and then in the same manner in 1% SDS/20 mM Na2HP04, pH 7.2, and subjected to autoradiography. Hybridisations with other probes used Ultrahyb (Ambion) hybridisation solution at 52 C and washes were performed according to the manufacturer's recommendations - 4 6 -2.3. Results 2.3.1. PCR screening and dot blot analysis In PCR reactions with the PR07 and PR04 primers, all the fungal isolates produced a -480 bp amplicon (Figure 2-1). Four of the O. minus isolates produced relatively strong amplicons while isolate 123-43-13 produced a weak amplicon. Two of the three C. resinifera isolates produced weak amplicons, but isolate 123-22-12 produced a strong amplicon. Other species gave nearly identical results between the representative isolates. Figure 2-2 shows that almost all of the Ophiostoma species produced a hybridisation signal in a dot blot analysis of their genomic D N A with the Prot6 probe. The few O. piceae, 0. minus and O. coronatum isolates that produced weak PCR amplicons also produced weak hybridisation signals. Most O. floccosum and O. setosum isolates produced a moderately strong hybridisation signal. For O. piliferum and A. pullulans only certain isolates produced a signal in the dot blot. A l l of the Ljptographium isolates produced a weak signal. C. resinifera strain 123-22-12 was the only C. resinifera isolate that produced a signal in the dot blot. - 4 7 -A Kb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 • 1 - > - -0.5 - > - " " " " * B Kb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0.5-*--~<*m wmm mmm mm mmm » Figure 2-1: PCR analysis results using the primers PR07 and PR04 on genomic D N A from 31 different fungal species. A) (1) X ladder standard (2,3,4) Ophiostoma piceae, (5,6) O. floccosum, (7,8,9) 0. piliferum, (10,11,12,13) Leptographium, (14,15,16) 0. coronatum. B) (1) X ladder standard (2,3,4,5,6) O. setosum, (7,8,910) O. / W » W J , (11,12,13,14)^.^//^/^, (15,16,17) C. resinifera. - 4 8 -1 2 3 4 5 6 7 8 9 10 11 § 1 • • # A • t B • • • • C * • • D Figure 2-2: A dot blot of 31 isolates of sapstaining fungi using the Prot6 Probe. 500ng of genomic D N A from 27 different isolates of sapstaining fungi were blotted, in alternate lanes, on to a Hybond-N+ membrane (Amersham). The membrane was hybridised overnight at 62C with the Prot6 Probe, (column #row #) species name: (1A,3A,5A) 0. piceae, (7A,9A) 0. floccosum, (11A,2B,4B) 0. piliferum, (6B,8B,10B,1C) Leptographium, (3C,5C,7C) 0. coronatum, (9C,11C,2D,4D,6D) 0. setosum, (8D,10D,1E,3E,5E) 0. minus, (7E,9E,1 IE) A.pululans, (2F,4F,6F) C. resinifera. - 4 9 -2.3.2. PCR-RFLP analysis In PCR-RFLP analysis of the PR07/PR04 amplicons with Taq 1 the generated band patterns could be divided into two groups (Figure 2-3). Group one consisted of lanes 9-12, representing the Leptographium spp. isolates, lane 24 the 0. minus isolate 58-4, and lanes 26-28 the C. resinifera isolates. For these isolates one Taq I cut site was present in the PR07/PR04 amplicons producing a band at ~360bp and one at ~100bp. Group two consisted of lanes 2-8 representing the O.piceae, 0. floccosum and 0. piliferum isolates, lanes 13-17 representing the 0. coronatum and 0. setosum isolates, lanes 19-23 that represented the O. minus and other 0. setosum isolates, and lane 25 the A., pullulans isolate. This group produced a variety of band patterns, all with multiple bands. The PR07/PR04 amplicons for the more common sapstaining species C. resinifera (125-214), heptographium (156-234), 0. minus (58-4a), 0. piliferum (156-112), 0. floccosum (55-1) and O.piceae (123-142) were cloned and twenty individual clones from each transformation were analysed by PCR-RFLP. Al l the individual C. resinifera, Ljptographium and 0. piceae clones produced the same band pattern; however, clones from 0. minus, 0. piliferum and 0. floccosum produced two distinct patterns. For 0. piliferum 18 of the clones produced the same pattern while two of the clones produced an alternative pattern. For 0. floccosum and 0. minus, both types of clones were equally represented. -50-bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 bp 18 19 20 21 22 23 24 25 26 27 28 w » *•#>* MM m i l Figure 2-3: I generated restriction digestion results, of P C R fragments generated with primers P R 0 7 and P R 0 4 from genomic D N A of 27 fungal isolates. Digests were performed at 65°C for 2 hours and then run on a 3% agarose gel for 45 minutes at 110 volts. (1) 1 kb X D N A ladder standard (2,3) O. piceae; (4,5) O. floccosum; (6,7,8) O. piliferum; (9,10,11,12) Leptographium; (13,14) 0. coronatum; (15,16,17) O. setosum, (18) 1 kb X D N A ladder standard (19,.20) O. (21,22,23,24) 0. OT/WWJ, (25) A.pullulans; (26,27,28) C. 500 - * • 1 •i 201-*-134 -*» -51 -2.3.3. Sequence analysis of PR04/PR07 PCR products The generated PR07 /PR04 amplicons for representative isolates from each of the twelve fungal species were cloned and sequenced. For 0. piliferum, 0. floccosum and 0. minus both amplicon types were sequenced. No intronic sequences were found in any of these sequences. Al l the obtained sequences had high similarity with known fungal protease K subfamily subtilases and contained the active site motifs D(S/T)G, G H G T H and GTSMA(S/T)P that are unique to subtilases. A dendogram based on an alignment of the inferred amino acid sequences from the obtained gene sequences and the amino acid sequences of other known fungal subtilases is presented in Figure 2-4. Three major branches were formed in the dendogram and delineated three major groups of protease K family subtilase sequences: the Cr, Oflocl and Opic groups. Seven of the sapstaining sequences grouped closely together in the Cr group. The rest of the sequences from sapstaining fungi were split between the Oflocl group and the Opic group. The Opic group shared only 30% amino acid identity with the other two groups, while the Oflocl and Cr groups shared approximately 50% amino acid identity with each other. Members within each group typically shared greater than 80% amino acid identity. The sequences of the amplicons from isolates that produced two amplicon types always belonged to separate groups, with one of the amplicon sequences always in the Cr group. - 5 2 -Figure 2-4: A n unrooted dendogram representation of the alignment of the inferred amino acid sequences of the P R 0 7 / P R 0 4 amplicon sequences obtained with the homologous regions from other known fungal subtilases. Group names are indicated above the first branch of the group. GeneBank accession numbers are shown in parentheses: Podospora ansenia alkaline protease (AF047689); A. nigerVBVC (M96758);.A nigerVEPB (LI9059);^4. nidulansVtxA (L31778);X ory^ae Alp (D10062); T. haryanum P rb l (M87518); T. album protease R (X56116); T. album endopeptidase K (AL445065); T. album protease T (M54901); A. fumigatus Alpl (AJ24315); A. fumigatus Alp (X66935); A. fumigatus alkaline protease (Z11580);M./>o<2<?Mpl (AF118126);M. anisopliaeVA (AB073345); Leptographium 156-234 Lr(AF413096); O. setosum 55-6-1 (AF413107); O. floccosum 387N Ofloc387N (AF413100); O. floccosum 55-1 Oflocl (AF413098); O. floccosum 55-1 Ofloc2 (AF413099); O. coronatum 68-2 (AF413097); O . ^ z ' ^ 123-142 Opic (AF413104) ;^ .^ / /« /««j 123-436 (AF413108); O. »w»»j 58-4 O m i n l (AF413101); O. W ^ J - 58-4 Omin2 (AF413102); O.piliferum 156-112 O p i l l l (AF413105); O.piliferum 156-112 Opil2 (AF413106); C. resinifera 125-214 Cr (AF413095); 0. novo-ulmi 16K (AF413103); O. Q142T (AF413094). -53 -70 58 69 56 83 62 99 331 Cr 100 81 O. novo-ulmi O. ulmi -O. floccosum 387N O. setosum O. minus Omin2 r C. nesinifera Leptographium sp. O. piliferum Opil2 Podospora ansenia Alkaline protease Aspergillus niger P E P C 96 •Aspergillus fumigatus Alp2 O. floccosum Ofloc2 100 95 -Tritirachium album protease R Oflocl 99 —Tritirachium album protease K -Metarhizium anisopliae Pr1 Tritirachium album protease T 83 87 100 I' Opic O. piliferum Opill O. floccosum Oflocl O. coronatum Magnaporthe poae Mp1 O. piceae 97 100 40 99 O. m/nus Ominl - Trichoderma harzianum Prb1 Apullulans 86 100 76 58 - A nidulans PrtA - A fumigatus Alp A n/'gerPEPD r A fumigatus Alkaline protease 99 I- A orj^ae Alp 0.1 - 5 4 -2.3.4. Screening of an 0. floccosum 387N genomic A, library Approximately 20,000 pfu from the genomic 0. floccosum 387N library were screened and ~20 A, phage clones that hybridised to the Prot6 probe were identified (Figure 2-5). PCR-RFLP was used to determine whether different subtilase genes had been obtained. The nine of A, phage clones tested produced the same band pattern (Figure 2-6). The D N A from one of the amplified clones was isolated and digested with different restriction enzymes and PCR was used to determine which of the bands contained the subtilase gene. Hind III produced the most suitable restriction pattern for subcloning (Figure 2-7). After ligation into pBluescript SK+ the insert was sequenced in the region surrounding the subtilase gene. Results of the analyses of the obtained sequence are presented in section 2.3.6. 2.3.5. RACE-PCR Sets of gene specific primers were designed from the obtained sequence information for opic from 0. piceae (123-142), Ir from ~Leptographium sp. (156-234), ^rfrom C. resinifera (124-214), oflocl from 0. floccosum (55-1), opill from O. piliferum (156-112) and opil2 from 0. piliferum (156-112). These genes were chosen because they are from commercially important species and because they are good representatives of each of the three identified subtilase groups. - 5 5 -Figure 2-5: A) primary and B) secondary library blots hybridised with the Prot6 probe Primary plates of an 0. floccosum 387N library in EMBL3 were plated at a density of ~2,000 pfu/plate onl50X15 mm petri plates, and secondary plates at 80-100 pfu/plate on 100X15 mm plates. Hybond N+ membranes were used to blot the plates and phage D N A was fixed by oven baking. Membranes were then hybridised to the Prot6 probe, as described in section 2.2.15. - 5 6 -B bp 1 2 3 4 5 6 7 8 9 10 iNMfl 298 —^-mm? Figure 2-6: PCR and PCR-RFLP analysis of nine X phage clones that hybridised to the Prot6 probe. A) PCR analysis results using the primers PR07 and PR04 on genomic DNA of the nine X phage clones: (1) X ladder standard (2-10) X phage clones 1-9. B) Taq I generated restriction digestion results, of PCR fragments generated with primers PR07 and PR04 from genomic DNA of nine X phage clones. Digests were performed at 65°C for 2 hours and then run on a 3% agarose gel for 45 minutes at 110 volts. (1) 1 kb X DNA ladder standard (2-10) X phage clones 1-9. -57-Figure 2-7: Genomic D N A from a X phage clone that hybridised to the Prot6 digested with different restriction enzymes. (1) 1 Kb ladder, (2) Hindlll and BamHl, (3) BamHl, (4) Hindlll. - 5 8 -PCR of the synthesized 3' and 5' cDNAs with the generated gene specific primers resulted in the amplification of appropriate amplicons (Figure 2-8). The bands ranged between 500 bp and 1 Kb, with the 5' amplicon slighdy larger than the 3' amplicon in each case. 0. piceae produced the smallest amplicons for both the 3' and 5' ends; otherwise, litde difference was seen in amplicon size between the species. No amplification product was obtained from the 5' reactions for oflocl or opill. This was likely due to the lack of a full-length transcript in these samples. Al l the generated bands were cloned and sequenced. 2.3.6. Analysis of full-length sequences A total of 2,057 bp were sequenced from ofloc387N. No introns were present in this sequence as the ORF was uninterrupted and aligned in an ungapped manner with other fungal subtilase sequences. Analysis of the region 5' to the A T G codon revealed the absence of a T A T A A box; however, several putative transcriptional elements were identified including a GC-box, a GCTCCS sequence and a C T N C N G sequence, which are alternative transcriptional activator sites [140]. Potential regulatory elements were also present including two GATAA-element binding sites and a possible Crea-like protem-binding site. For era total of 2,341 bp was sequenced and about 500 bp of sequence 3' to the T G A stop codon was obtained. A potential A A T A A A polyadenylation site was identified in this highly T rich region. From opil2 1,511 bp of sequence was obtained and a start metMorune was located. From Ir 1,536 bp of sequence was obtained, and although an A T G codon was identified, it was unlikely that this codon represented the true start site, as a protein lacking a - 5 9 -Kb 1 2 3 4 5 6 7 8 9 10 2.0 ""•» :-J 1.6 0 . 5 — * Figure 2-8: RACE PCR results of selected isolates of staining fungi using gene specific primers and universal primer mix. (1) 1 kb A. D N A ladder standard; (2) 3' amplification of O.piceae cDNA; (3) 5' amplification of O.piceae cDNA; (4) 3' amplification of Lep/ographium cDNA; (5) 5' amplification of Leptograpbium cDNA; (6) 3' amplification of O. floccosum cDNA; (7) 5' amplification of O. floccosum cDNA; (8) 3' amplification of C. resinifera cDNA; (9) 5' amplification of C. resinifera cDNA; (10) 5' amplification of O. piliferum opi/2 cDNA. - 6 0 -PRE region and with a very short PRO region would be produced. Transcriptional start sites were not found for the opic, opill, and ofloc2. Additional information for these sequences is shown in Table 2-2. Table 2-2: Analysis of obtained full-length sequences Sequence Total ORF PRE PRO Mature Mature Pi Name length of sequence obtained (bp) (a.a.) (a.a.) (a.a.) enzyme (a.a.) enzyme (kDa) ofloc387N 2057 440 15 135 290 30.0 6.8 cr 2341 504 15 139 350 30.0 7.1 Ir 1536 420 - 70 326 33.0 7.1 opic 1122 419 - 105 314 31.7 6.4 ofloc 1593 290 - - 290 29.4 6.1 opill 1234 285 - - 285 33.0 7.0 opil2 1511 503 15 128 364 36 6.4 The generated full-length sequences shared at least 50% identity at a D N A level in regions coding for the active enzyme. An alignment of the inferred amino acid sequences of all the full-length sequences obtained is shown in Figure 2-9. Al l the sequences contained the active site serine within the highly conserved motif SGTSMA(T/S)PH that is shared by all protease K subfamily subtilases. Cr group sequences contained the active site serine in the sequence SGTSMASPH, while sequences from the Opic and Oflocl groups contained the sequence SGTSMATPH. One exception to this rule was noted as protease T contained the active site serine in the same sequence as the Cr group enzymes despite being from the Oflocl group. The active site aspartic acid was present within the conserved sequence -61 -c.res HKRrmsummpAiirrirrTHro^ 70 L E P ASG GIAKG 8 0 . H . O 3 HHRFITrLALAVGASAAPAFSTETIHG]>AAPILSSAWAEEVPGAYinCFT<DHVlJEDKASDHHSUVQTIH(JT 70 0 . p i l 2 MKSAVLMSLASVAVAAPSFTTGTVHGDAAPILASVWTEAIPNSYIVKFKHHVNEKD V3THHS 62 O . p i l l i 0 . f l o e i O . p i c AVVTrQSTRALKDSAAVDTESHLSWVTNLHF^SLSARNTAGIEKTYH ISMUSA S3 C . r e s UVKSFRR ATRSATSSCA3AAYWQVQTRSSAVS3TLTPLGEFLGYAGHFI>I>SIIELVRKIPGVEVIHR 137 L e y KIWFFRR SQG FPLPHUMSFSVMQAISHILSSS WSAGIRHSSTIPAAVCRP S8 HVKTFPEKEDEAGPRMOAUPRSAPLRGRLSAGLKHTYKDWHGLLGYAGHFDDSIIEQVTISS 133 WVQSLHDQGEEKR LELRRRGHDFGILSGVKHTFKIGETFKGYAGQFDDELIEQLPPHPDVEYIEK 127 1 C . r e s L e p O . f l o 3 0 . p i l 2 O . p i l l O . t l o c O . p i c C . r e s L e p O . f l o 3 0 . p i l 2 O . p i l l B.jj'iS O . t l o c ( O . p i c GCGNYSSIVERGSYDAAADKKTEEPEAADVLGAVSDIEKPLSLT-GCGNYSSIVERGSYDAAADKKTEEPEAAD 504 420 4 4 0 GCSNYSSILEQGSYKVKSSKWSKLDIEALEKAIEHDLHIVSGQIVKGAESLASK 503 PSA 285 PSA 290 419 Figure 2-9: Alignment of the full-length subtilase sequences obtained. The sequences aligned are 0.flo3: O. floccosum 387N Ofloc387N (AF413100); O.flo: O. floccosum 55-1 Oflocl (AF413098); O.pic: O.piceae 123-142 Opic (AF413104); O.pill: O.piliferum 156-112 Opill (AF413105); 0.pil2: O.piliferum 156-112 Opil2 (AF413106); C. res: C. resinifera 125-214 Cr (AF413095); Lep: Leptographium 156-234 Lep (AF413096). Homologous sites are shaded in dark grey and similar sites in light grey. - 6 2 -D(S/T)G for all of the sequences. There was intragroup variation in the residue -5 from the active site aspartic acid. At this site, Cr group sequences contained an apartic acid residue, while Oflocl group sequences contained a cysteine residue, and Opic group sequences contained a tyrosine residue. The active site histidine was present within the motif G H G T H in all of the sequences. The opic sequence from 0. piceae contained three insertions of 11, 6 and 5 amino acids near hypothetical loop structures, as determined by PHD_sec; as well, Ir from Leptographium contained one significant insertion of 15 amino acids. Otherwise, all the sequences were almost identical in length with the only substantial differences being in the length of 3' terminal extensions after the active site serine motif. A dendogram based on an alignment of the inferred amino acid sequences of the mature enzymes with other fungal protease K family subtilases is shown in Figure 2-10. The topology of the tree generated agreed very closely with the tree generated from the partial sequence information, confirming the segregation of the sequences into three groups. A dendogram based on the alignment of the PRE-PRO regions shows the segregation of the sequences into two major groups (Figure 2-11). Prl from M. anisopliae grouped with members of the Oflocl group in this analysis, although in other analyses it grouped in the Opic group. This could indicate that only two major types of PRE-PRO regions are present in fungal subtilases those for intracellular subtilases and those for extracellular subtilases. More sequences are, however, needed to verify this statement. 93 70 66 I A7 100 88 99 AA 63 -100 1 oo r C. resinifera 65 100 57 L 100 'Leptographium sp. - O . floccosum 387N —Podospora ansenia Alkaline protease — O. pilifemm Opil2 -Aspergillus nigerPEPC 100 Aspergillus fumigatus Alp2 —Tritirachium album protease R -Tritirachium album protease K -Metarhizium anisopliae Pr1 Tritirachium album protease T •O. piliferum Opill — O. floccosum Oflocl Magnaporthe poae Mp1 100 I -Trichoderma harzianum Prb1 O. piceae 53 A nidulans PrtA A n/grerPEPD -A fumigatus Alp [A fumigatus Alkaline protease 100 IA oryzae Alp 0.1 Figure 2-10: An unrooted dendogram representation of the alignment of the inferred amino acid sequences of the full-length sequences from other known fungal subtilases. GeneBank accession numbers are shown in parentheses: P. ansenia alkaline protease (AF047689); A. % r P E P C (M96758);yl nigerVEPB (L19059);.A nidulansYttk (L31778);^. ory^ae Alp (D10062); T. harxiannmVtb\ (M87518); T. album protease R (X56116); T. a/tow endopeptidase K (AL445065); T. album protease T (M54901); A. fumigatus Alp2 (AJ24315); A. fumigatus Alp (X66935); A. fumigatus alkaline protease (Z11580); M / J O ^ M p l (AF118126); Af. anisopliae Pr l (AB073345); Leptographium 156-234 Lep (AF413096); O. floccosum 387N Ofloc387N (AF413100); O. floccosum 55-1 Oflocl (AF413098); 0./>z'^ 123-142 Opic (AF413104); 0. piliferum 156-112 Opill (AF413105); O. piliferum 156-112 Opil2 (AF413106); C. fRfwjfos 125-214 Cr (AF413095). 80 | 55 45 95 99 47 62 I - 6 4 -• O. floccosum 387N • C. resinifera -Podospora ansenia Alkaline protease — O . piliferum Opil2 -Aspergillus niger PEPC —Aspergillus fumigatus Alp2 Tritirachium album protease K Tritirachium album protease R 100 100 -Trichoderma harzianum Prb1 -Metarhizium anisopliae Pr1 89 58 - A nigerPEPD -A nidulans PrtA -A fumigatus Alp IA fumigatus Alkaline protease 1 0 0 ' A oryzae Alp 0.2 Figure 2-11: An unrooted dendogram representation of the alignment of the inferred arnino acid sequences of the PRE-PRO regions of subtilase sequences. The sequences aligned are: P. ansenia alkaline protease (AF047689); A. nigerVEVC (M96758);yl nigerVEVD (LI9059); A. nidulansVttA (L31778);X oryzae Alp (D10062); T. hanQanum Vih\ (M87518); T. protease R (X56116); T. a/tow endopeptidase K (AL445065); A. fumigatus Alp2 (AJ24315); A. fumigatus Alp (X66935); A. fumigatus alkaline protease (Z11580);M anisopliae Pr l (AB073345); O. floccosum im* Ofloc387N (AF413100); O. piliferum 156-112 Opil2 (AF413106); C fRnwg&ra 125-214 Cr (AF413095). - 6 5 -2.3.7. Southern blot analysis Figure 2-12 shows that the opill (Oflocl group) probe produced a single band of approximately 2.3 kb in the 0. piliferum lane, a weak band of similar size in the 0. piceae lane, and a band of approximately 2.8 kb in the 0. floccosum lane. The presence of a single band in each of these lanes indicated that these genes were present as single copies. 0. piceae had not been shown to contain a gene from the Oflocl group indicating the presence of a previously unidentified subtilase gene in this species. No band was found in either the C. resinifera or the Leptographium sp. lanes, indicating that these species do not contain genes with sufficient sequence identity to the probe. The opil2 (Cr group) probe produced a band of about 5.5 kb in both the 0. piliferum and the 0. floccosum lanes. The presence of a single band under the conditions tested indicated that these genes were present as single copies. No bands were present in the 0. piceae, C. resinifera or Leptographium sp. lanes at the hybridisation temperature used. The probe generated from the C. resinifera subtilase gene hybridised with all species using low stringency conditions, except for 0. piceae. 0. piliferum, 0. ulmi and 0. novo-ulmi only produced one band, of a relatively moderate intensity that indicated the presence of a single copy of these genes. 0. floccosum, C. resinifera and Leptographium, all produced two bands. In 0. floccosum both bands were of equal intensity, indicating the possible presence of two copies of this gene. In both C. resinifera and Leptographium the second band was much - 6 6 -A Kb 1 2 3 4 5 4.0 -*> 3 . 0 -2.0 1.6-*-B Kb 1 2 3 4 5 4.0--* 30"* m& I C 1 2 3 4 5 Kb 12.0-» 6.0-* 5.0 - * H 4.0-* ^ 1 2 3 4 5 6 7 Kb 3.0-* 2 0 ~*" * # * 1 . 6 - » ^ • Figure 2-12: Southern blots of genomic D N A of sapstaining fungi. lOug of fungal genomic DNA's were digested as described in section 2.2.10, unless otherwise noted, and probed A) using a probe derived from the opic subtilase gene (1) O. piliferum 156-112, (2) O. piceae 123-142, (3) O. floccosum 55-1, (4) Leptographium 156-234, (5) C. resinifera 125-214. B) using a probe derived from the opi/1 gene (1) O. piliferum 156-112, (2) O. floccosum 55-1, (3) O. piceae 123-142, (4) Lep/ographium 156-234, (5) C. resinifera 125-214. C) using a probe derived from the opi/2 gene (1) O.piliferum 156-112, (2) 0. floccosum 55-1, (3) O.piceae 123-142, (4) Leptographium 156-234, (5) C. resinifera 125-214. D) lOug of fungal genomic DNA's were digested Hindlll and probed using a probe derived from the ^rgene (1) O. floccosum 55-1 (2) O. piceae 123-142, (3) 0. piliferum 156-112, (4) C. resinifera 125-124 (5) Leptographium 156-234 (6) O. (7) O. ulmi. - 6 7 -weaker, mdicating the possible presence of a second subtilase type in these species. No hybridisation signal was produced by 0. piceae in either of the blots with the Cr group probes, suggesting that this species does not contain a gene from this group. The opic (Opic group) probe produced two separate bands, of approximately 2.3 kb and 2.8 kb respectively, in the O. floccosum lane indicating that this gene was present as two unlinked copies. This gene probe also produced a single band at around 3 kb in the 0. piceae lane indicating the presence of a single copy of this gene. No other lanes showed bands, suggesting that other staining species did not contain similar genes. - 6 8 -2.4. Discussion PCR and dot blot screening results indicated that subtilase genes are ubiquitous in sapstaining fungi; however, sequence variations were apparent. PCR-RFLP with TaqX was used to better assess these variations. This enzyme was chosen as it had a potential cleavage site in a region lacking sequence conservation, and was likely to produce divergent band patterns. Al l the isolates from the C. resinifera, 0. piceae and Leptographium species produced a band pattern indicating the cleavage of a single PR07/PR04 amplicon into two fragments. Isolates from other Ophiostoma species produced band patterns that indicated that two or more separate PR07 /PR04 amplicons were being cleaved. PCR-RFLP of individual clones confirmed the presence of two subtilase genes in all of the Ophiostoma species tested, with the exception of 0. piceae. All the sequences obtained shared a high degree of similarity with known fungal subtilases and their inferred amino acid sequences exhibited motifs common to this group of enzymes. A dendogram based on the alignment of the inferred amino acid sequences formed three groups of sequences. The Opic group shared about 30% sequence similarity with the other two groups, which shared 50% sequence similarity with each other. Sequence similarity was > 80% within the groups. The Cr group included an intracellular A., fumigatus protease sequence that is involved in spore formation, PEPC that was reported to play a housekeeping role and an intracellular subtilase involved in vegetative incompatibility in P. anserina [103] [99] [110]. In this - 6 9 -analysis all the intracellular, but no extracellular, subtilase sequences were included in the Cr group. This suggested that other Cr group members are also intracellular. Phylogenetic analysis revealed that the pre-pro regions of intracellular and extracellular subtilases are distincdy different; reflecting their importance in protein localisation. The Oflocl group included the extracellular subtilases protease T, K and R, as well as Pr1 from M. anisopliae that is involved in the cuticle degradation of the exoskeleton of host insects [122]. Other members of this group may also be extracellular and possibly involved in pathogenicity. Similarly, sequences from the Opic group are likely extracellular and possibly involved in pathogenicity. Five extracellular Aspergillus subtilase sequences clustered together in the Opic group, including a subtilase from A. fumigatus that plays a role in pathogenicity [103] [101] [106] [107] [100]. Also in this group was Prbl from T. hartnanum that is involved in mycoparasitism [114]. In order to confirm the segregation of these groups, RACE-PCR and library screening techniques were used to obtain full-length sequences from several subtilase genes. The dendogram, created from an alignment of the full-length sequences, showed the same grouping found with the partial sequences, supporting the segregation of the sequences into three groups. The inferred amino acid sequences for the full-length genes shared common secondary and tertiary structural elements with protease K. The lengths of the encoded open reading frames (ORF's) varied from 440 a.a. for Ofloc387N to 503 a.a. for Opil2. Other fungal subtilase genes have ORFs that vary substantially in length. Several are relatively short, - 7 0 -between 273 a.a to 295 a.a., mcluding the Alp genes horn. A. fumigatus and A. ory^ae, and PEPD from A. niger [104] [107] [99]. Another group ranges from 402 a.a. to 409 a.a., and includes Prb1 from T. har^ianum and Mp1 from M. poae [114] [111]. Two ORF's of 527 a.a. and 531 a.a. have been identified in Psp2 from P. brassicae and PspA from P. anserina, respectively [93] [110]. An ORF of 790 a.a. was identified in Prt1 from P. carinii [109]. The lengths of the encoded PRE-PRO regions remained relatively constant. Variations in the lengths of the sequences occurred in regions coding for the mature enzyme, which ranged from 285 a.a. for Opill to 364 a.a for Opil2. The major differences in lengths were mainly due to 3' terminal extensions, as for Opil2, Lr and Cr, or to insertions, as in Opic and Lr. 3' terminal extensions have been reported to play a role in vacoular targeting [93]. The significance of the insertions is unknown but they usually occur in loops and most indels act to increase or decrease the relative size of these regions [88]. The obtained ofloc387N sequence contained no introns. Introns are common in fungal subtilase genes and can range in number from one, in Psp2 from P. brassicae, to seven, as in prt1 from P. carinii [93] [109]. Intron size can vary gready as well, ranging from 51 bp up to 227bp, as in Psp2 from P. brassicae [93]. Southern analyses indicated that most Ophiostoma species are likely to contain Oflocl group homologues, but homologues from the Opic group have only been identified in some Ophiostoma species. 0. piceae was the only sapstaining species tested for which no Cr group gene was identified. C. resinifera and Leptographium sp. do not seem to contain any subtilase - 7 1 -genes aside from the Cr group genes identified. 0. floccosum was found to contain two copies of an Opic group gene and possibly a Cr group gene as well. Most other fungal subtilases have been reported as single copies, however, in M.poae, Mp1 was present as at least two copies, and in P. carinii,prt1 was present in many copies throughout the genome [100] [135] [111] [109]. - 7 2 -2.5. Conclusions The work presented in this chapter shows that subtilases were essentially ubiquitous in sapstaining fungi, as was expected considering the important nutritional role they are hypothesised to play in this group of fungi. Intraspecies variations in these sequences for most of the species tested were discovered by the use of a variety of techniques. Sequence analysis revealed that three major groups of subtilases were present in fungi. The full-length sequences of seven subtilases, with at least one representative from each of the three groups, were determined. Most of the subtilase genes were present as single copies, however, multiple copies of some of the genes were found. The work presented in this chapter provided a foundation upon which further analyses, such as regulational analyses, could be performed to determine the physiological roles of the encoded enzymes. -73 -Chapter 3 : Regulational analysis and heterologous expression of subtilase genes from sapstaining fungi2 3.1. Introduction In the previous chapter three groups of fungal subtilases were described. It was not, however, possible to determine if these groups had physiologically distinct roles. Two common methods for determining a gene's possible function are to study its regulation and to analyse the substrate specificity and biochemical properties of the encoded protein. This chapter describes both methods to determine the possible roles of three representative subtilase genes. The first objective of this work was to study the regulation of the three representative genes. Previously, the regulation of protease genes from several fungal species has been studied in an attempt to clarify their physiological roles. For example, the regulation of members of the secreted aspartic protease (SAP) gene families from R oryyae and C. albicans have been well characterised [121] [97] [130] [129]. In both species, several of the genes were regulated in a similar fashion while others were regulated in a unique manner. The regulation of several fungal subtilase genes has also been studied. This includes PR1 from M. anisopliae, prbl from T. hartnanum and AaSP2 (torn A. astaci, which all responded to induction by host tissues, implying that they play a role in nutrient acquisition and pathogenesis [122] [114] 2Portions of this work have been submitted for publication: Hoffman, B., Breuil, C. 2003. Analysis of the Distribution and Regulation of Three Representative Subtilase Genes in Sapstaining Fungi. Submitted to Fungal Genetics and Biology - 7 4 -[115]. Two other fungal subtilase genes,psp2 from P. brassicae and AaSP1 £tomA. astaci were shown to be constitutively expressed and were reported to play a housekeeping role [93] [115]. The second objective of this work was to perform a biochemical analysis of the proteins encoded by the representative genes. In order to carry out these studies sufficient quantities of each of the proteins were required. Heterologous expression systems are often used to produce large quantities of proteins, and have been employed to produce several different fungal subtilases. For example, an alkaline protease from A. fumigatus was expressed in E. coli as a fusion protein with a his-tag, and the alp genes from Ammonium chrysogenum and A. ory^ae were heterologously expressed in S. cerevisiae [141] [117] [107]. This chapter describes the regulation by pH, available nutrients and culture age of opi/1 from the Oflocl group, opi/2 from the Cr group and opic from the Opic group. These genes each represented one of the three identified subtilase groups. Both multiplex RT-PCR and Northern analyses were used to determine the expression levels of the genes. The total extracellular proteolytic activity and growth of the cultures were also monitored and correlated to the expression of the genes. As well, the genes were heterologously expressed in E. coli to produce and purify sufficient quantities of each of the encoded subtilases to determine their substrate specificities. -75 -3.2. Materials and Methods 3.2.1. Fungal and Bacterial strains and growth conditions Fungal isolates 0. piliferum 156-112 and 0. piceae 123-142 were obtained from the U.B.C. Wood Science Department (Vancouver, Canada) and stored as described in section 1.2.1 [20]. Pre-cultures were grown at 20-23 C for 5 days in malt extract broth (ME) (Oxoid). Cultures were filtered through 3 layers of sterile cheesecloth and the yeast and spore cells collected by centrifugation at 3,000 x g and re-suspended at 1X108 cells/ml in water and left overnight at 20 C. One ml of the cell suspension was used to inoculate 20 ml of minimal media at the appropriate pH containing specific carbon and nitrogen source (Table 3-1), and incubated at 20 C for the appropriate time. Cultures biomass was determined by weighing mycelia that were filtered through a 70 um nylon cell strainer (Falcon) and dried. 3.2.2. Protease assays Cultures were filtered through a 70 um nylon cell strainer and culture filtrates collected. Proteolytic activity was determined by adding 50 ul of each culture filtrate to 100 ul of 2 mg/ml succinylated casein (Pierce) in 50 mM borate buffer pH 8.5, 50 ul of buffer was - 7 6 -Table 3-1: C o m p o n e n t s o f art if icial m e d i a usee i n mul t ip l ex R T - P C R and n o r t h e r n analyses M e d i a # M e d i a contents* Ini t ia l p H G r o w t h t ime N u t r i t i o n a l Regu la t i on E x p e r i m e n t s 1 W a t e r 7.0 48 hours 2 0 . 1 % N H 4 C 1 , 2 % G l u c o s e 7.0 48 hours 3 1% B S A , 0 . 1 % N H 4 C 1 , 2 % G l u c o s e 7.0 48 hours 4 1 % B S A , 0 . 1 % N H 4 C 1 7.0 48 hours 5 1 % B S A , 2 % G l u c o s e 7.0 48 hours 6 1 % B S A 7.0 48 hour s p H Regu la t ion E x p e r i m e n t s 1 1 % B S A 4.0 48 hours 2 1% B S A 5.0 48 hours 3 1% B S A 6.0 48 hours 4 1% B S A 7.0 48 hours 5 1% B S A 8.0 48 hours T i m e Cour se E x p e r i m e n t s 1 1 % B S A 7.0 0 hours 2 1% B S A 7.0 12 hours 3 1% B S A 7.0 24 hour s 4 1% B S A 7.0 48 hour s 5 1 % B S A 7.0 96 hour s *A11 media con ta ined B - m e d i u m as descr ibed by A b r a h a m [142] - 7 7 -added in control reactions. Reactions were incubated for 2 hours at 37 C. 50 ul of trinitrobenzenesulfonic acid was then added and the reactions further incubated at room temperature for 20 minutes to allow colour development which was measured at 450 nm. Reactions and controls were done in triplicate. To determine the amount of enzymatic activity in the samples the means of the absorbance readings of the appropriate controls were subtracted from the means of the absorbance's of the reactions. One unit of protease activity was defined as the amount of enzyme required to produce an absorbance of 1. 3.2.3. Purification of Genomic D N A , Total RNA and mRNA Purification of fungal genomic D N A was done using the method described by Kim et al [17] with the addition of a two-stage phenol chloroform extraction prior to D N A precipitation by isopropanol. For the purification of total RNA mycelia were first disrupted while frozen by drilling as described in Kim et al [17]. The total RNA was then extracted using a Trizol based method (Invitrogen). - 7 8 -3.2.4. Multiplex RT-PCR Contaminating DNA's were removed from total RNA by adding 1 uL of 1 M MgCl 2 (BDH Chemicals), and 10 units of DNase I FPLCpure (Amersham Pharmacia Biotech) and incubating at 37 C for 30 minutes. Reverse transcription (RT) was carried out using Superscript II (Invitrogen) with a poly T 2 0 primer. cDNA's were then purified using a QIAquick Cleanup Kit (Qiagen). cDNAs were stored at —20 C for short term storage, or — 80 C for longer times. Multiplex RT-PCR reactions were performed using the p-tabulin specific primers T8 and T l 0 in conjunction with the appropriate gene specific primers (Table 3-2). The T8/T10 primer pair was designed to span an intron in the [3-mbulin gene, producing a ~550 bp amplicon from genomic D N A and a ~250 bp amplicon from cDNA, which allowed the easy detection of contaminating D N A . Reactions used a 40:1 ratio of j3-tubulin primers to gene specific primers because this ratio was empirically shown to provide successful amplification of both genes. These reactions used 40 amplification cycles with an annealing temperature of 56 C. Al l reactions were repeated three times. Bands were quantified from each of the triplicate reactions using Sigma Scan Pro (SPSS Inc.). The ratio of the gene specific amplicon to the ^-tubulin amplicon for each lane was determined from these intensities. The ratios from the triplicate reactions were averaged to obtain the mean ratio for the samples. - 7 9 -Table 3-2: List of Synthetic Oligonucleotides Target Primer Name Sequence Tubulin gene T8 G A C C G A A G A T G A A G T T G T C G TIO A C G A T A G G T T C A C C T C C A G A C 26s rDNA NL1 G C A T A T C A A T A A G C G G A G G A A A A G NL2 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 opi/2 wP2-3 C C T T G G G G T C T C G C T C G T A T C T C C wP2-6 G T T G G G G G T G T C C T T G G G C A G opill wPl-1 C C C T G G G G C A T C T C G C G C wPl-4 G G T T G C C G T T G A A A G C C A G C T opic wPic-t T C G G G C T C T A C C T G G G G C C T G G G wPic-2 G C G G A A G G T G A T G G C A G C A G G G C 3.2.5. Northern Analysis Probes were prepared using the wP2-3/6 amplicon for the opi/2 gene, the wPl-1 /4 amplicon for the opill gene, and the wPic-1/2 amplicon for opic gene, and the N L 1 / N L 2 amplicon for the 26s region of the rDNA. Each probe was labelled with 50 pCi (32P)a-dCTP (Amersham Pharmacia Biotech) using Ready-To-Go Labelling Beads (Amersham Pharmacia Biotech). Unincorporated nucleotides were removed with G-50 micro columns (Amersham). For Northern analyses 20 pg of total RNA was loaded per lane and separated in 1.4 % formaldehyde agarose gels. Total RNA was then transferred to Zeta-Probe GT blotting membranes (Bio-Rad) using downward capillary transfer with 10 x SSC, and fixed to the membranes by baking for 2 hours at 80 C. Membranes were hybridised with the appropriate probe using Ultrahyb solution (Ambion) at 50 C. Two room temperature washes were first performed using 2 x SSC, 0.1% SDS for 5 min, and then two more washes at 50 C were - 8 0 -performed using 0.1 x SSC, 0.1% SDS for 15 minutes each. Probes were stripped by adding boiling 0.1% SDS which was then allowed to cool to room temperature. 3.2.6. Heterologous Expression The primers pairs wPl - l /wPl -4 , wP2-3/wP2-6, and wPic-l/wPic-2, were used to amplify the 0. piliferum opill gene, the 0. piliferum opil2 gene, and the O. piceae opic gene, respectively. The obtained amplicons were cloned in frame into the p B A D / T O P O thiofusion expression vector (Invitrogen) and positive clones were screened by PCR to determine the orientation of the insert. Appropriate clones were then sequenced to ensure that the subtilase genes were present in the correct reading frame and that no sequence alterations had occurred during cloning. Expression tests were conducted using 10 ml of overnight cultures induced with a series of arabinose concentrations ranging from 2x10"5 % to 2 xlO"1 % for 4 hours at 37 C. Cells were centrifuged at 3 000 x g for 10 min and the supernatants and pellet analysed by sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) using a 10-20% linear gradient gel. The expressed enzymes were purified from 100ml cultures grown to an O D of ~0.5 then induced with 0.02% arabinose and grown for another 4 hours. Cells were then collected at 3000 x g for 10 min, and resuspended in solubilization buffer (6M guanidine-HCL, 20 mM NaP0 4 , 500 mM NaCl, pH 7.8). After insoluble debris was removed, the enzyme mixture was loaded onto a ProBond Resin matrix column (Invitrogen). The expressed his-tagged proteins were then purified using first a linear pH gradient with solubilization buffer from pH 7.8 to pH 6.0 and then an elution step at pH 4.5. The purified proteins were analysed by SDS-PAGE using a 10-20% linear gradient gel. - 8 1 -Proteins were allowed to renature by buffer exchange using dialysis with 50 mM Tris-HCl buffer, pH 7.5. Protease activity assays used 3.0 mg Azocoll suspended in 2.0 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 10 mM CaCl 2 and 0.2 M NaCl that was incubated at 37 C for 15 min then 0.1 mg of purified protein was added to the azocoll suspension and incubated at 37 C for 4 hrs with shaking. Proteolytic activity was determined by measuring the absorbance of the filtrate at 520 nm. - 8 2 -3.3. Results 3.3.1. Nutrient Regulation of the Expression of Representative Subtilase Genes In multiplex RT-PCR reactions using the opill primers an 834 bp amplicon was produced in all the lanes corresponding to cultures that contained BSA (Figure 3-1). A p-mbulin amplicon of about 280 bp was produced in all the RT samples and a —500 bp p-tubulin amplicon was produced in the genomic control lane. These bands were present in all subsequent multiplex RT-PCR analyses. The p-tabulin primers used span —220 bp of intronic sequence and the absence of a -500 bp amplicon in the RT samples indicated the absence of contaminating DNA's. The opill/tubulin primer ratios were not significantiy different for the lanes in which an opill amplicon was produced. In the Northern analysis with the opill probe a —1.6 kb hybridisation signal was detected in all of the lanes corresponding to cultures grown in media containing BSA. The size of this transcript agreed with the size of the opill gene. The blot was stripped and then probed with a 26s rDNA specific probe which was used as a control for RNA loading between the lanes. This probe produced a signal in all of the lanes in this and subsequent analyses. An 837 bp amplicon was produced in all of the lanes from multiplex RT-PCR reactions using the opil2 gene specific primers (Figure 3-1). The opil2/tubulin primer ratios were not significantiy different for any of the lanes. As well faint —1.6 kb hybridisation signals were produced by the opil2 gene probe in all of the lanes in the Northern analysis. -83 -opM op/72 opic Glucose NH,i BSA + + - + -+ + + - - - + + + + + _-«-_ + + + - -- + + + + + - L + -1 • Gene Specific * Primers W! <fa/M -- — : : Tubulin Primers -*• . » • « • « — - « • « * » " » 0*,'',°oJi,. ro.ro r0 r0 >j 'J 'j >j *, *, *, ro, ro 'J *j V 'J >J B Glucose + + - + _ + + _ + -NHi BSA - + + + 4-+ + + -- + + + + I + + + + Gene Specific Probe 26s probe i f v • m 0 4- 4- + - n i I * - - • ^ * Figure 3-1: Analysis of the regulation of the opi/1, opi/2 and opic genes by exogenous nutrient sources. Fungal yeast cells or spores were inoculated into media supplemented with the nutrients indicated above the lanes and grown for two days. A) Multiplex RT-PCR results using the gene specific primers for the opi/1, opi/2 and opic genes and the tubulin specific primers (Table 2). The mean ratio of the gene specific amplicon to the tubulin amplicon for each lane with the standard deviation in brackets is indicated below the lanes. L: lkb D N A ladder (Invitrogen); lanes 2-7: cDNA from cultures grown in media containing the nutrients indicated above the lane; + Genomic control multiplex PCR using the appropriate gene specific primers and the tubulin specific primers with 50 ng of genomic D N A ; - no RT negative control. B) Northern Analyses of 20 pg of total RNA from cultures grown in media containing the nutrients indicated above the lane with opi/1, opi/2 and opic gene probes. The blots were stripped and then reprobed with a 26s rDNA probe as a control for RNA loading levels. - 8 4 -An -856 bp amplicon was produced using the opic gent specific primers in the multiplex RT-PCR reactions from all of the cultures grown in media that contained BSA in the absence of either glucose and/or ammonia. The opic/tubulin primer ratios were not significantly different in these lanes. Reactions from cultures grown in media that contained both ammonia and glucose, or did not contain BSA, did not produce an opic gene amplicon. In the Northern analysis using the opic gene probe, hybridisation signals of -1.6 kb were only present in lanes corresponding to cultures grown with BSA in the absence of either glucose and/or ammonia. Figure 3-2 shows that the total extracellular proteolytic activity increased in 0. piliferum cultures grown in the presence of BSA, but both carbon and nitrogen repression affected the level of activity produced. Cultures grown in BSA alone produced 5 times higher levels of proteolytic activity than cultures grown in only glucose and ammonia. Cultures grown in media containing both BSA and glucose with or without ammonia had 60% less activity than those grown in BSA alone. Cultures grown in media containing BSA and ammonia had 35% less activity. Significant differences in the biomass produced were not noted in any of these media types. Total extracellular proteolytic activity of O. piceae cultures grown with BSA as the only nitrogen and carbon source, was approximately 14 times greater than in glucose and ammonia. Cells grown in media containing ammonia and BSA produced approximately - 8 5 -1 E N LU A 0.12 0.1-0.081 0.061 0 .04 -0.02 J 0 Glucose N H 4 B S A B Nutrient Type E c uu Glucose NH4 B S A Nutrient Type Figure 3-2: Extracellular Proteolytic Activity in response to different nutrients. A) O. piliferum and B) O. piceae were grown in media containing the nutrients indicated for two days, the cultures were then filtered and 50 pi of the filtrate assayed for total proteolytic activity. Assays were done as described in section 3.2.2. - 8 6 -half the proteolytic activity of cells grown with BSA alone. This indicated that nitrogen repression played a strong role in inhibiting the proteolytic activity produced by 0. piceae. Glucose seemed to have little effect, and glucose/BSA cultures produced levels of proteolytic activity that were as high as for cultures grown in BSA alone. 3.3.2. pH Regulation of the Expression of Representative Subtilase Genes In the multiplex RT-PCR reactions an opill amplicon was produced from cultures that were buffered to pH 6, 7 and 8 but not to pH 4 or 5 (Figure 3-3). The amplicon from the pH 7 culture was stronger in intensity, producing an opi/1 /tubulin primer ratio 50 % greater than those obtained from either pH 6 or pH 8. In the Northern analysis a -1.6 Kb hybridisation signal was also detected in the lanes corresponding to cultures that were buffered to pH 6, 7 and 8, but the signal was more intense for pH 7 and pH 8. Gene specific primers for opi/2 produced amplicons in all of the multiplex RT-PCR reactions, and the opi/2 probe produced weak hybridisation signals in all of the lanes in the northern analysis (Figure 3-3). The amplicons produced roughly the same opi/2/tubulin primer ratios and the bands in the northern analysis, although very weak, were of approximately the same intensity. - 8 7 -opM Gene Specific Primers 500bp> Tubulin Primers - * opic 4 5 6 7 8 L + % f*r \ •ii •i ~ii r± >, f, ro r0 r0 fQ •r o B PH 4 5 6 7 8 4 5 6 7 8 Gene Specific Probe 26s probe 4 5 6 7 8 •w?*^ 'HBP V". Figure 3-3: Analysis of the regulation of the opill, opil2 and opic genes by exogenous pH. Fungal yeast cells or spores were inoculated into BSA media buffered to the pPTs indicated above the lanes and grown for two days. A) Multiplex RT-PCR results using the gene specific primers for the opill, opil2 and opic genes and the mbulin specific primers (Table 2). The mean ratio of the gene specific amplicon to the tubulin amplicon for each lane with the standard deviation in brackets is indicated below the lanes. L: lkb D N A ladder (Invitrogen); lanes 2-7: cDNA from cultures grown in BSA media buffered to the pH's indicated above the lanes; + Genomic control multiplex PCR using the appropriate gene specific primers and the tubulin specific primers with 50 ng of genomic D N A ; - no RT negative control. B) Northern Analyses of 20 pg of total RNA from the cultures with opill, opil2 and opic gene probes. The blots were stripped and then reprobed with a 26s rDNA probe as a control for RNA loading levels. - 8 8 -Gene specific primers for opic only produced amplicons in multiplex RT-PCR reactions from cultures grown at pH 7 and pH 8 (Figure 3-3). The opic/tabulin primer ratios were not significantiy different in either of these lanes. Hybridisation signals were produced from cultures grown at pH 6, 7, and 8; however, the signal from the pH 6 culture was very weak. The total extracellular proteolytic activities of 0. piliferum cultures increased by approximately 40% from pH 4 to pH 5 (Figure 3-4), and then dropped by roughly 60% from pH 5 to pH 6. The level of activity was maintained at roughly the same level in the cultures buffered to pH 7 and 8. This trend also occurred in O. piceae. Cultures grown at pH 5 produced about 40% greater activity than those grown at pH 4 and approximately 60% greater than those buffered to pH 6. The levels of activity produced were similar in cultures grown at pH 6, 7 and 8. Significant differences in the biomass produced were not noted in any of these media types. 3.3.3. The Effect of Fungal Culture Age on the Expression of Representative Subtilase Genes With the exception of the initial (time zero) time point, the opill gene specific primers produced amplicons in the multiplex RT-PCR reactions from cultures grown for all time points tested (Figure 3-5). The opill/tabulin ratios produced were approximately the same for each of the time points. A —1.6 kb hybridisation signal was also detected for these time 89-A >s .ti .s 0) E c UJ 6 pH 6 PH Figure 3-4: Extracellular Proteolytic Activity in response to exogenous pH. A) 0. piliferum and B) 0. piceae were grown for two days in BSA media buffered to the pETs indicated, the cultures were then filtered and 50 pi of the filtrate assayed for total proteolytic activity. Assays were done as described in section 3.2.2. - 9 0 -Gene Specific Primers 500bp> Tubulin Primers - * B time (h) Gene Specific Probe 26s probe L 1 opill opH2 opic 0 12 24 48 96 + - L 0 12 24 48 96 + - L 0 12 24 48 96 + -__ ! m ~~ '**" *"* f., i> f. a . .,;„ «* * w -H** ' ° n ?s> *s> / 0 0 rf> . O _ 5 r°, r°, ••ii •ii * ro r0 r0 r0 rb -ii "ii Si * 0 12 24 48 96 0 12 24 48 96 >^ ^ . * ^ 12 24 48 96 i n a n Hi W mm Figure 3-5: Analysis of the regulation of the opill, opil2 and opic genes by fungal culture age. Fungal yeast cells or spores were inoculated into and grown for the times indicated above the lanes. A) Multiplex RT-PCR results using the gene specific primers for the opill, opill and opic genes and the tubulin specific primers (Table 2). The mean ratio of the gene specific amplicon to the mbulin amplicon for each lane with the standard deviation in brackets is indicated below the lanes. L: lkb D N A ladder (Invitrogen); lanes 2-7: cDNA from cultures grown for the time points indicated above the lane; + Genomic control multiplex PCR using the appropriate gene specific primers and the mbulin specific primers with 50 ng of genomic D N A ; - no RT negative control. B) Northern Analyses of 20 pg of total R N A from cultures grown in media containing the nutrients indicated above the lane with opill, opil2 and opic gene probes. The blots were stripped and then reprobed with a 26s rDNA probe as a control for RNA loading levels. - 9 1 -points in the northern analysis, but the 12 hour sample produced a significandy fainter signal. The opi/2 gene specific primers produced amplicons in the multiplex RT-PCR reactions, and the opi/2 probe hybridisation signals in the northern analysis, for all of the time points tested (Figure 3-5). Roughly the same opi/2/tubulin primer ratios were produced by all of the samples and the bands in the northern analysis were of approximately the same intensity. In the multiplex RT-PCR reactions with the opic gene specific primers an amplicon was produced from cultures grown for 24 hours or longer (Figure 3-5). The opic/tubulin amplicon ratio was roughly the same for each of these time points. The northern Hybridisations with the opic probe also produced signals from cultures grown for 24 hours or longer and the intensities of the signals produced were roughly equivalent. The total extracellular proteolytic activity of 0. piliferum cultures increased significandy during the first 12 hours then stayed roughly the same as the culture aged further (Figure 3-6). The biomass of the cultures lagged behind the proteolytic activity, increasing the most between 12 and 24 hours of growth and then continuing to increase as the culture aged further. In 0. piceae the total extracellular proteolytic activity increased linearly in cultures grown for 0 hours to 48 hours. No further increase in activity was noted for cultures grown for longer periods of time. Again the biomass of the cultures lagged behind the proteolytic activity and increased linearly only after 12 hours of growth. Figure 3-6: Extracellular proteolytic activity and growth in response to culture age. A) 107 yeast and/or spore cells of 0. piliferum and 0. piceae were grown in BSA media for the times indicated and 50 pi of the filtrate assayed for total proteolytic activity as described in section 3.2.2, and B) the growth of the 0. piliferum and 0. piceae cultures was measured as described in section 3.2.1. -93 -3.3.4. Heterologous Expression Induction experiments of the expression of Opil l , Opil2, and Opic from their respective pBAD/TOPO thiofusion expression vectors were performed using a series of arabinose dilutions. The results of these experiments indicated that expression of the proteins increased up to an arabinose concentration of 0.02% after which no further increase was noted (Figure 3-7). The supernatants of disrupted cells contained very low concentrations of the expressed proteins and the majority of the expressed proteins were insoluble. A denaturing buffer was used to solubilize the lysed cellular material for the purification of the enzymes. Purification of the his-tagged proteins was carried out using a Probond Resin matrix column. The bulk of the proteins were eluted in the first 50 fractions using a linear pH gradient in the elution buffer from pH 7.8 to pH 6 (Figure 3-8). SDS-PAGE analysis of these fractions indicated that they contained little to none of the heterologously proteins (Figure 3-9). The remaining proteins were eluted by dropping the pH of the elution buffer to pH 4.0. SDS-PAGE analysis of these fractions indicated that the majority of the protein in these fractions was the heterologously expressed protein of interest, and showed that the fusion proteins were approximately 50 kDa in size (Figure 3-9). Approximately 1.2-2.4 mg of each of the fusion proteins was purified from the initial 83-120 mg of insoluble cellular -94 -1 2 3 4 1 2 3 4 1 2 3 4 Figure 3-7: SDS-PAGE analysis of insoluble protein from E. coli cultures induced with an increasing concentration of arabinose. The opill, opil2, and opic genes were cloned into pBAD/TOPO thiofusion vectors under the control of an arabinose promoter. These vectors were transformed in E. coli, which were induced with increasing concentrations of arabinose, Lane 1 0.0002%, Lane 2 0.002%, Lane 3 0.02%, Lane 4 0.2%, to produce the heterologously expressed proteins A) Opill , B) Opil2, and C) Opic. -95-0.35 =3 .Q C o d) u— o X 64 Fraction # Figure 3-8: Representative purification profile of the Opil2-tmoredoxin fusion protein. 100ml cultures of E. coli harbouring an Opil2 expression vector were induced with 0.02% arabinose. The cells were then pelleted and then resuspended in solubilization buffer (6M guanidine-HCL, 20 mM NaP0 4 , 500 mM NaCl, pH 7.8). The resulting protein mixture was loaded onto a ProBond Resin matrix column (Invitrogen), and expressed his- tagged proteins were then purified using a linear pH gradient of solubilization buffer from pH 7.8 to pH 6.0 (dashed line) and then an elution step at pH 4.5. The A 2 g 0was monitored to determine the protein concentration in the fractions (solid line). - 9 6 -Figute 3-9: SDS-PAGE analysis of different fractions obtained from the purification of A) Opill , B) Opil2, and C) Opic. 100 ml cultures of E. colt harbouring the appropriate expression vectors were induced with 0.02% arabinose The cells were then pelleted and then resuspended in solubilization buffer (6M guanidine-HCL, 20 mM NaP0 4 , 500 mM NaCl, pH 7.8). The resulting protein mixture was loaded onto a ProBond Resin matrix column (Invitrogen), and expressed his- tagged proteins were then purified using first a linear pH gradient with solubilization buffer from pH 7.8 to pH 6.0 for 57 fractions and then an elution step at pH 4.5for fractions 58-73. Lane 1: solubilized protein mixture before addition to the column, Lane 2: Fraction 15, Lane 3: Fraction 30, Lane 4: Fraction 45, Lane 5: Fraction 65. - 9 7 -protein, indicating that the fusion proteins were produced as approximately 1.4-2% of the insoluble cellular material. Buffer exchange was then performed in order to allow the renaturation or refolding of the purified proteins. Enzymatic analysis showed that the fusion proteins had no detectable proteolytic activity. To ensure that the absence of activity was not due to the presence of the thioredoxin fusion group, enterokinase treatment was used to remove the N-terminal leader sequence. After this treatment the 37kDa proteins were purified and assayed; however, no activity was detected. - 9 8 -3.4. Discussion Gene regulation often occurs in a hierarchical manner. For proteases, pH is often the dominant factor, as was reported for members of the SAP gene families in C. albicans and R. ory^ae [121] [124] [132] [135] [130]. pH was also the dominant factor in the regulation of opill and opic and these genes were only expressed in cultures grown at pH 6 or higher. pH regulatory systems typically prevent the production of extracellular enzymes and other proteins in conditions where they would be unable to function [124]. Subtilases are inactive at acidic pH's, having activity optima at neutral to alkaline pH's. The total extracellular proteolytic activity of the 0. piliferum and 0. piceae cultures was higher at slightly acidic pH, indicating that aspartyl or other acid proteases that are typically induced at an acidic pH may have been produced [131] [134] [121]. In P. roqueforti p H mediated repression of the aspartyl protease gene aspA works indirectiy, as no inducing peptides are produced at pH's where ASPA is inactive [132]. On the other hand, several fungal species including A. nidulans, S. cerevisiae and Yarrow lipolytica have been shown to contain pH regulatory pathways [124]. In these systems ambient alkaline pH triggers a signalling pathway that modifies a ^ rC-like transcriptional activator, making it susceptible to proteolysis [124]. The proteolysis of this transcriptional activator removes its inhibitory domain allowing it to affect the activation of alkaline expressed genes and the repression of acid expressed genes. In Y. lipolytica this system is required for the production of alkaline proteases [124]. opill and opic were likely regulated by similar pH mediated - 9 9 -systems and not simply by the absence of inducing peptide because proteolytic activity in the cultures was high at acidic pH's. Proteases are also commonly regulated by nutritional factors and are often repressed by carbon, nitrogen or sulphur and/or induced by exogenous protein sources [143] [121-124]. In this work, opill was induced by exogenous protein but was not significantly repressed by either ammonia or glucose. Exogenous protein sources have been shown to induce other proteases including sapl-3 in K ory^ae and sap2 in C. albicans [121] [130]. In M. anisopliae the expression ofprl was induced specifically by chitin [74]. Gene induction by exogenous protein occurs by the stimulation of GAL4 family transcriptional activators by peptides produced by the degradation of the protein source [74]. The identity of activators specific to protease production and whether this stimulation occurs directly or indirecdy is unknown. The level of extracellular proteolytic activity in 0. piliferum cultures did, partially, correlate with the expression of opill and the activities of cultures grown with BSA increased substantially. The level of total extracellular proteolytic activity produced was affected by both glucose and nitrogen repression and the production of other extracellular proteases regulated by these factors also contributed to the level of activity determined. Although opic was also induced by BSA, the removal of either nitrogen or carbon repression was required for the expression of this gene. For sapl, sap2 and sap3 from R oty^ae, and acpl of S. sclerotiorum, the absence of either carbon or nitrogen repression was shown to allow expression, which increased in the presence of exogenous protein [121] [134]. Nitrogen - 100 -repression in fungi acts through the repression of GATA-binding transcriptional activators by glutamine [74] [73]. In the absence of favoured nitrogen sources intracellular levels of glutamine are to low to prevent the binding of these transcriptional activators to the promoters of nitrogen regulated genes. The binding of these activators in co-ordination with the binding of pathway-specific activators, as described above, allows the transcription of specific genes. Regulation by carbon sources works through the production of global-acting repressor proteins [80-82]. In the absence of favoured carbon sources these repressor proteins are not produced, allowing the induction of genes by pathway-specific induction signals. The expression of opic was probably regulated by similar systems in O.piceae. Although induction by BSA was the determining factor in the level of proteolytic activity produced by O. piceae, glucose did repress the levels of activity produced. Nitrogen repression had little effect suggesting that proteases other than Opic contributed to the extracellular activity of the O. piceae cultures. Induction of opill began witxiin 12 hours and opic within 24 hours of inoculation of the cultures into BSA. This indicated that both opill and opic responded directly to induction by BSA or by peptides derived from BSA. In this experiment these genes were not induced by the autolysis of older dying mycelia. Levels of extracellular proteolytic activity mirrored the expression of the genes, and maximum activity was reached within 12 hours of growth for O. piliferum and within 24 hours for O. piceae. The cultures were unable to grow substantially until high enough levels of proteolytic activity were produced to provide sufficient amounts of required nutrients to the growing cultures, after which the biomass of the cultures increased linearly. - 101 -The expression of opt/2 did not vary over time and did not respond to regulation by available nutrient sources or to exogenous pH. Several researchers have reported the presence of constitutively expressed proteases mcluding sap9 and sap10 from C. albicans, the subtilase genepsp2 from P. brassicae and AaSP1 from A. astaci [130] [93] [115]. Phylogenetic analysis showed that Opil2 and other Cr group proteins, such as Psp2 and AaSPl, are very similar to PEPC in A. niger, which is a vacuolar protease involved in protein turnover [144]. This suggested that opil2 is a constitutively expressed housekeeping gene encoding a protein that may be involved in the activation of other enzymes. To perform biochemical analyses on the proteases encoded by these genes it was necessary to purify large amounts of each of the proteins. Purification of a serine protease from O. floccosum 387N culture filtrate has been carried out. This technique was, however, inefficient and time consuming [70]. A heterologous expression system was developed in order to obtain sufficient quantities of the enzymes. To remove the need for post-translational processing PCR primers were designed to amplify the mature coding regions of the genes without a pre-pro region or C-terminal extension. The amplified PCR fragments were cloned into the p B A D / T O P O thiofusion expression vector which adds both a his-tag for easy purification and a thioredoxin tag that enhances the solubility of the expressed protein. In expression tests inducer concentrations greater than 0.02% did not increase protein production and the majority of the expressed proteins were insoluble. The heterologously expressed proteins were purified using a nickel sepharose column under denaturing conditions with a linear pH gradient in the elution buffer. After renaturation, the enzymes had no detectable proteolytic activity, even after the removal of the N-terminal tags. This - 102-was probably due to the improper folding of the expressed proteins. Several studies have shown that P R O regions of serine proteases are necessary to guide their folding into the proper conformation [94] [95]. -103 -3.5. Conclusions The expression of opill, which was used as a representative of the Oflocl group, was induced by BSA, but not affected by nitrogen or carbon repression. The expression of this gene, under inducing conditions, was regulated by pH, indicating that pH repression was able to override induction by BSA. The expression of opill occurred within twelve hours when cultures were grown in inducing conditions. The expression of opic, which was used as a representative of the Opic group, was also induced by BSA but required the removal of either nitrogen or carbon repression to be expressed. The expression of opic was also repressed in acidic conditions, again indicating the ability of the pH regulatory pathway to override induction signals. The expression of opic began within 24 hours of growth in inducing conditions. On the other hand, opill was expressed in all the conditions tested. Each of the proteins encoded by these genes was heterologously expressed in E. coli. The expressed proteins were insoluble and were purified under denaturing conditions. After renaturation of the proteins they had no detectable proteolytic activity. The purified proteins that were obtained could be used to develop antibodies, which would allow the localisation of these subtilases in mycelia. These data could help provide information on the potential roles these subtilases may play in the physiology of the fungi. - 104 -The roles of these genes in the growth of sapstairiing fungi in wood are still unclear. In this work, opi/2, which was used as a representative of the Cr group, was expressed under all conditions tested indicating the possible housekeeping nature of this gene. On the other hand, opi/1 and opic responded to exogenous nutrient sources indicating that they play a role in nutrient acquisition. This, however, remains to be confirmed. - 105 -Chapter 4 : The physiological roles of opill and opic in sapstaining fungi3 4.1. Introduction The previous chapter showed that opill and opic responded to exogenous nutrient sources incucating that the enzymes they encode may play a role in nutrient acquisition. In this chapter gene disruption is used in an attempt to determine the roles of these two genes. Gene disruption has been used to determine the physiological roles of other fungal proteases, including SAP (1-6) in C. albicans [127-129]. Disruption of SAP1, SAP2, and SAP3 attenuated but did not eliminate virulence in murine and guinea pig models indicating that none of the three genes was the primary virulence factor in these models [128]. In an estrogen-dependent rat vaginitis model SAP2 was the dominant virulence factor, indicating that SAP2 contributes to the pathogenicity of C. albicans in vaginitis [129]. A strain of C. albicans containing a triple-deletion of SAP4, SAPS, and SAP6 was also constructed. This mutant was unable to grow using BSA as a nutrient source and exhibited lower virulence [127] [130]. It was suggested that one or more of these genes was required for the proper activation of SAP2 [127]. 'Portions of the work presented in this chapter have been submitted for publication: Hoffman, B., Breuil, C. 2003. The Roles of the Two Subitilase Genes, opill and opi/2, in Ophiostomapi/iferum's Growth on Wood. Submitted to Molecular Plant-Microbe Interactions. - 1 0 6 -Subtilase genes from A. fumigatus and A. awamori have also been disrupted using homologous recombination strategies [102] [108]. The disruption of a subtilase gene horn A fumigatus did not affect the pathogenicity of the isolate tested, and the authors indicated that this gene was not the predominant protease expressed in mouse lung [102]. Disruption of a subtilase in A. awamori produced transformants with a reduced ability to degrade heterologously expressed protein [108]. In order to perform gene disruptions, a transformation system for the target species is required. Classically, fungal transformation has been achieved by first digesting fungal cell walls using hydrolytic enzymes to produce protoplasts. DNAs are then introduced into the generated protoplasts by electroporation or with poly ethylene glycol (PEG) [145-147]. DNAs then integrate into fungal chromosomes via double crossover events [148] [149]. O. piceae, Ophiostoma quercus and O. ulmi have been transformed using a P E G mediated system, however, the transformants generated typically contained multiple copies of the integrated D N A [150] [151]. For gene disruption it is desirable to have a single integration site to avoid any uncertainty about the observed phenotype arising from the disruption of non-target genes. Recently, Agrobacterium tumefaciens has been used to transform a number of fungi including Mycosphaerella graminicola, Fusarium oxysporum, Fusarium circinatum,A. bisporus, Coccidiodes immttis and others [152-157]. This system has also been used to transform two species of sapstaining fungi, O.piceae and C. resinifera [158] [159]. In this system, T-DNA is transferred into intact fungal cells from an A. tumefaciens Ti plasmid. The T-DNA then integrates into a chromosome of the host fungus. Typically, transformants with only a single copy of the - 107 -integrated D N A are generated and the frequency of recombination at homologous regions may be increased over other techniques [153] [160]. These fmdings makev4. tumefaciens mediated transformation (ATMT) particularly useful for gene disruption studies. This chapter describes the attempted gene disruptions of opic and opill that were carried out in order to prove their physiological roles. First the transformation of O. piceae and 0. piliferum using A T M T with the disruption vectors is described. The obtained transformants were screened for their growth and for their ability to produce extracellular proteolytic activity. Southern blot analysis was used to determine if the selectable H P H marker gene had recombined into the opill locus of representative transformants. Multiplex RT-PCR was used to ensure that disrupted genes were unable to produce a wild type transcript. -108 -4.2. Materials and Methods 4.2.1. Fungal and Bacterial strains and growth conditions Fungal isolates O.piliferum 156-112 and O.piceae 123-142 were obtained from the U.B.C. Wood Science Department (Vancouver, Canada) [20]. Fungi were grown at 18-23 C for 2-5 days on M E A (Oxoid Ltd) plates, which were overlaid with cellophane (BioRad) when mycelia were used for D N A extraction. Fungi were stored as previously described (Section 2.2.1) Transformants were grown on 2 % S M A (Difco) plates to assess their extracellular protease activity. The growth of the transformants on BSA and BSA/NH 4 C1/Glucose media was measured as described in Section 3.2.1., and on wood as described by Fleet [49]. Three wood blocks were infected for each transformant. The E. coli strain T O P 10 (Invitrogen) was used in all cloning experiments and grown on L B agar plates containing 100 ug/ml of ampicillin or no antibiotic, as was appropriate. A. tumefaciens GV3103 was grown, stored, and transformed as described by Pikaard [161]. 4.2.2. Ergosterol Analysis - 109-Ergostetol extraction and analysis was performed by Rod Stirling according to the method of Pasanen [162]. Four grams of freeze-dried wood was refluxed for one hour in 100 m L of 10% K O H in methanol. The solution was allowed to cool, filtered and washed with 50 m L of distilled water. The methanol solution was extracted with hexane (2x100 mL) and rotovaped to dryness. The samples were transferred to Reacti-vials with hexane and the cholesterol internal standard was added. The samples were silylated using 150 uL of bis-ttimethylsilyl-trifluoroacetamide and 45 uL of pyridine at 60°C for 45 minutes. Finally, the samples were made up to 1.5 m L with hexane and analysed by G C / M S . A Saturn 3800 series G C / M S with a 30 m DB-5 column was used for the analysis. Injector temperature was 290°C. 1 uL of sample was injected with a split ratio of 20. Helium carrier gas flow rate was 1 m L / m i n . Ion source temperature was 220°C. Ionization energy was 70eV. A solvent delay of 6 minutes was applied. Column temperature was ramped at 10°C/min from 170°C to 290°C and held for 12 minutes. Identification of the analytes was based on their retention times and mass spectra. The cholesterol-trimethylsilyl internal standard and ergosterol- trimethylsilyl eluted at 16.8 minutes and 17.8 minutes respectively. A blank sample of hexanes, pyridine and bis-trimetiiylsilyl-tjrifluoroacetamide did not exhibit any peaks in this region. Integrated peak areas were determined using the Satview software. A calibration curve was developed based on the ratio of ergosterol and cholesterol peak areas. Based on this curve the concentration of ergosterol in the samples was determined. -110 -4.2.3. Disruption Vector construction Restriction digests and ligations were done according to standard protocols [137]. To construct the disruption vectors the wPl - l /wPl -4 and the wPIC-l/wPic-2 primer pairs were used to amplify portions of the opill and opic genes, respectively. The generated amplicons were cloned into the pCR2.1 TOPO TA vector (Invitrogen) to generate the vectors pwPl and pwPic. The hygromycin B resistance cassette was amplified from the vector pCB1004, a gift from Louise Glass (University of California at Berkeley, USA), using the primers Thga-1 (5 ' -GTC-GAC-GTG-GAG-GTAATA-ATT-GAC-GAC-GAT A-3') andThga-2 (5'-GTC-GAC-AAC-GTT-TrC-CAA-TGA-TGA-GCA-C-3') [163]. These primers amplified 1.6 Kb of the hygromycin cassette with the addition oiAccl restriction sites on either side of the amplicon. This amplicon was TOPO cloned, then excised with Acc\ and ligated into Accl digested pwPl and pwPic to generate the vectors pwPl-HPH and pwPic-HPH. The pwPl-HPH vector contained 589 bp (residues 13-602) of opill flanking the 5' end of the H P H expression cassette and 244 bp (residues 603-847) of opill flanking the 3' end. The pwPic-HPH vector contained 184 bp (residues 349-533) of opic flanking the 5' end of the H P H expression cassette and 490 bp (residues 713-1203) of opic flanking the 3' end. The created disruption cassettes were excised from these vectors by digestion with Hindlll IXbaX and ligated into Hindis /Spel digested pCAMBIA-0380 (Cambia) to create the vectors pCwPl-HPH and pCwPic-HPH (Xbal and Spel have cohesive compatible ends). An overview of this process, using the construction of pCPl -HPH as an example, is presented in Figure 4-1. - I l l -Figure 4-1: The construction of the disruption vector pCwPl-HPH. The H P H gene was excised from pHPH-TOPO by digestion with Accl, and subsequently cloned into the Accl site of pwPl to generate pwPl-HPH. The wpl-HPH cassette was then excised from this plasmid by digestion with Hindlll/Xbal and directionally cloned into a Hindlll/Spel digested pCambia-0380 vector, resulting in the disruption vector pCwPl-HPH. - 112-4.2.4. A. tumefaciens-mediated transformation Yeast like cells of 0. piliferum and 0. piceae were grown in 2% malt extract at 20 C, with shaking at 200 RPM, for 5 days. Cells were filtered through three layers of cheesecloth, and centrifuged at 2,000 X g for 5 minutes. The obtained cells were washed twice with sterile distilled water and resuspended at 1 X 108 cells/ml. The plasmids pCwPl-HPH and pCwPic-HPH were transformed into A. tumefaciens and the resulting strains grown at 26 C for 2 days in rriinimal medium (MM) with 50 pg/ml kanamycin while shaking at 300 RPM [164]. Cells were then added to induction medium (IM) supplemented with 200 uM acetosyringone to an O D 6 0 0 of 0.15 [165]. The cells were grown for 6 hrs at 26 C with shaking at 300 RPM. Equivalent volumes (10 ml) of the generated A. tumefaciens cells and fungal cells were mixed. 1 ml of this mixture was spread onto cellophane sheets (BioRad) layered on IM agar plates supplemented with 200 pM acetosyringone. The cell mixture was grown for 5 days at 20 C. The cellophane sheets with cells were then transferred to M E A plates supplemented with 300 g/ml hygromycin, 200 pM cefataxime, and 100 U g / m l of moxalactum to select for transformants and to kill the remaining^, tumefaciens cells. Transformants were transferred to M E A supplemented with 300 g/ml hygromycin, and single spore isolations were subsequently performed. - 113 -4.2.5. Purification of Genomic D N A and Total RNA (TRNA) Purification of fungal genomic D N A and total RNA were performed as described in section 3.2.3. 4.2.6. Southern Analyses 1 pg of the wPl - l /wPl -4 amplicon was digested with No/1, to remove the 5' end of the amplicon, purified, and then labelled with 50 pCi (32P)a-dCTP (Amersham) using Ready-To-Go Labelling Beads (Amersham). Unincorporated nucleotides were removed with G-50 micro columns (Amersham). For southern analysis 10 pg of genomic DNAs were digested with 10 units of Not\ for 16 hours and separated on 1.0 % agarose gels at 60 volts for 8 hours. DNA's were then transferred onto Zeta-Probe GT blotting membranes (Bio-Rad) according to the manufacturer's recommendations. Hybridisation and washing conditions were as described in section 2.2.10. 4.2.7. Multiplex PCR and RT-PCR - 1 1 4 -Multiplex PCR reactions were performed using the rDNA specific primers NL1 and NL2 in conjunction with wPl-1 and dPl-2 (5 ' -CCT-CAT-GGT-CGG-TGT-GGC-AAA-GTA -3'). Reactions used a 40:1 ratio of the wPl-1 / dPl-2 primer pair to the N L 1 / N L 2 primer pair, as this ratio was empirically shown to provide successful amplification of both genes. Reactions used 40 amplification cycles with an annealing temperature of 56 C and were repeated three times. The primer rPl-2 (5 ' -GGT-GGC-GCC-GAC-AGT-GCA-GAC-G-3') was used also used. For multiplex RT-PCR's, contaminating DNA's were removed from the total RNA by adding 1 pL of 1 M MgCl 2 (BDH), and 10 units of DNase I FPLCpure (Amersham) before incubating at 37 C for 30 minutes. Reverse transcription (RT) was carried out using Superscript II (Invitrogen) with a poly T 2 0 primer. cDNA's were purified using a QIAquick Cleanup Kit (Qiagen). cDNAs were stored at —20 C for short term storage, or —80 C for longer times. PCR reactions with the wPl - l /wPl -4 and NL-1/N1-2 primer pairs were performed as described above. 4.2.8. Protease assays Cultures were filtered through a 70 pm nylon cell strainer and culture filtrates collected. Proteolytic activity was determined as described in section 3.2.2. - 115 -4.3. Results 4.3.1. Transformation of 0. piceae and 0. piliferum Transformants appeared on M E A supplemented with 300 pg/ml hygromycin plates after 5-7 days. For O. piliferum approximately 20-30 transformants were obtained from each plate, while for O. piceae 5-10 transformants were obtained. The transformations were repeated twice, with similar results, giving a transformation efficiency of approximately 50 transformants/107 spores for O.piliferum and 15 transformants/107 spores for O.piceae. Control transformations with the pCambia vector had similar transformation efficiencies. A total of 200 O. piliferum and O. piceae transformants were transferred to new selective media and all continued to grow. 4.3.2. Phenotypes of mutants generated on M E A and SMA Two hundred O.piliferum and O.piceae transformants and the wild type strains were then inoculated onto SMA and M E A plates without hygromycin selection. For 0. piceae none of the transformants produced a decreased skim milk clearing zone. Three transformants appeared to cause crevasses in the media, and one produced an abundance of conidia. PCR screening of the 200 transformants using purified genomic D N A showed that the disruption - 116-vector had not integrated into the opic locus in any of the transformants. No further analyses were done on these mutants. For 0. piliferum, several groups of phenotypes were obtained (Figure 4-2). On M E A , fourteen transformants produced few to no aerial hyphae, had a lower growth rate and were much darker in colour relative to the wild type. Eighteen transformants produced increased aerial hyphae and had a salt-and-pepper coloration pattern. Three transformants were grey with abundant whitish aerial hyphae. Eleven transformants were brown and velvety in appearance. On SMA plates several phenotypes were also observed (Figure 4-3). Fight transformants produced a larger clearing zone and were blacker than the wild type. Thirty-five transformants produced little to no noticeable clearing zone on skim milk agar. Several had an increased linear growth rate but grew at a decreased density, producing large colonics with uneven borders. Others were albino on SMA although they appeared normal on MEA. Many transformants that produced a reduced clearing zone also had altered cultural morphologies. Five showed no morphological differences with the wild type on SMA or MEA. Representative transformants were then single spore isolated and re-grown on selective media and subsequendy on M E A and SMA to reconfirm their phenotypes. All maintained their ability to grow on hygromycin after three successive rounds of plating and reproduced the same phenotypes as previously noted, indicating that these transformants contained stably integrated copies of the disruption cassette. -117-WT J 2 3 Figure 4-2: Wild type O. piliferum (WT) and 6 selected transformants grown on M E A for two weeks. Transformants 39, 19, 21, 34, 30 and 23 were inoculated onto M E A plates and their growth rates and morphologies were compared to the wild type strain after two weeks of growth at 25 C. - 118-23 Figure 4-3: Wild type O. piliferum (WT) and 6 selected transformants grown on SMA for two weeks. Transformants 39, 19, 21, 34, 30 and 21 were inoculated onto SMA plates and their growth rates and morphologies were compared to the wild type strain after two weeks of growth at 25 C. Clearing zones are indicated by a white bar. -119-4.3.3. Southern Blot Analysis and Multiplex PCR Southern analysis was used to determine if the disruption cassette had integrated into the opill locus of six transformants that provided a wide cross section of the types of transformants obtained. These included, three that produced litde to no noticeable clearing zone in the skim milk agar (isolates 21, 34 and 23) and three that produced a normal clearing zone in the skim milk agar (isolates 19, 30 and 39). First Notl was used to digest the genomic DNAs from the transformants and from the wild type. Noil cuts opill near the 5' end of the gene. This cut site was present in the 5' ends of both the wild type gene and opill in the disruption cassette (Figure 4-4). A Notl cut site was also present between the 3' end of opill and the left T-border of the disruption construct. This site would be inserted into the host genome during a random integration event but would be lost during homologous integration mediated by the 5' and 3' opill segments. These analyses used a Notl digested opill probe, which removed the 5' end of opill. This reduced the number of bands in the blot and made the analysis clearer. In the southern blot, the wild type produced a band of ~5.2 Kb (Figure 4-5). This band was produced by Noil cutting the 5' end of opilll and at a site 3' to the gene. Transformants 39, 19 and 30 produced a band of similar size as well as a band of ~2.2 Kb. This band was produced from Notl cutting at the 5' and 3' ends of the disruption cassette. The presence of these two bands indicated that the disruption cassette had randomly inserted into the genomes of these transformants. - 120 -opill Probe rP1-2 trpC HPH \MP1-4dP1-2 I I I I I I I I I I I I I I I l l l l I M , , , , , ! , , | | I , , , 800 1.0kb 1.2kb 1.41* 1.6kb 1,8kb ' " " I " ' I " i I I ' nml 2.0kb 2.2kb 2.4kb 2.6kb 2725 D i s r u p t e d opill trpC 111.1111 II 11 11)1 I i I I II u u It 1111 II If It II II 11 II 11 11 11 II HPH 200 600 800 1 M b 1.2Kb 1.4kb 1.6kb 1.! l i i l * I l l l l i i n i l i M . I n . . . . , , / 2.0kb 2.21* 2.4kl> 2.6kb 2.8kb 2884 R a n d o m l y I n t e g r a t e d opill d i s r u p t i o n c a s s e t t e Figure 4-4: Schematic representation of the wild type opill gene, a hypothetical disrupted opill gene, with the hygromycin expression cassette inserted into the gene, and a randomly integrated disruption cassette. Relevant primer sites are shown, as are the locations of Notl restriction sites and the location of the opill probe. - 121 -K b W T 3 9 19 21 3 4 3 0 2 3 Figure 4-5: Southern blot analysis of 6 selected transformants and the wild type 0. piliferum with an opill probe. 10 pg of genomic D N A from each of the transformants and the wild type (WT) was digested with Not! and run on a 1% agarose gel for 8 hrs. The D N A was transferred and hybridised with a Noil digested wPl- l /wPl -4 amplicon probe. Hybridisation and washes were performed as described in section 4.2.6. The blot was exposed for 2 days. - 122 -Transformant 21 produced both a ~2.2 Kb band and a ~6.2 Kb band. The ~6.2 Kb band could have resulted from the integration of the disruption cassette into the wild type opill locus. This should have produced a ~6.6 Kb band, since the ~1.4 Kb hygromycin expression cassette would have been added to the ~5.2 Kb band produced by the wild type. This suggested that a portion of either the disruption construct, opill, or of the surrounding genome was deleted during integration. Transformants 34 and 23 produced only the ~6.2 Kb band mdicating that the disruption cassette had specifically integrated into the opill locus in these transformants and that some of the sequence was lost during integration. PCR and multiplex PCR were used to evaluate what portion of the sequence was lost during the integration of the disruption cassette into the opill locus. Al l the transformants were hygromycin resistant and produced an expected size amplicon in reactions using the Thga-l/Thga-2 primer pair, which spans the entire hygromycin expression cassette (data not shown). This indicated that the loss did not occur within the hygromycin expression cassette. The primer dPl-2, which has no priming site in the disruption construct, was used with wPl-1 to determine whether the 5' and 3' ends of opill were still intact (Figure 4-6). In these reactions the wild type and transformants 39, 19, 21 and 30 all produced both a NL-l / N L - 2 amplicon and a wPl - l /dP l -2 amplicon. Transformants 21, 34 and 23 only produced a N L - l / N L - 2 amplicon, indicating that either the wPl-4 or dPl-2 priming site was lost in these transformants. -123 -WT 39 19 21 3 4 30 2 3 - v e w p 1 - 1 / d p i - 2 N L 1 / NL2 Figure 4-6: Multiplex PCR using opill specific and ITS specific primer pairs with the genomic D N A of 6 selected transformants and wild type 0. piliferum. 50 ng of genomic D N A of each of the transformants and the wild type (WT) was amplified using the NL1/NL2 and wPl - l / dP l -2 primer pairs in multiplex PCR reactions using a hybridisation temperature of 56 C with 40 cycles of ampUfication. L: 1 Kb D N A ladder (Life Technologies), -ve: 20 ng of pCwPl-HPH. - 124-PCR reactions were then performed using the primer rPl-2, which binds just 5' to the hygromycin gene in the disruption cassette, with wPl-1. These reactions produced amplicons of equal size for all the transformants and the wild type (Figure 4-7). This indicated that wPl-1 primer site was not lost and that 5' end of opill was still present in the targeted transformants. As the wPl - l /dPl -2 primer pair did not produce an amplicon in these transformants the lost portion of sequence must have been deleted from the 3' end of opill during the homologous integration of the disruption cassette. 4.3.4. Multiplex RT-PCR To determine whether opill was expressed in the targeted transformants multiplex RT-PCR was performed. In these reactions, all the transformants and the wild type produced an NL-l / N L - 2 amplicon (Figure 4-8). Only the wild type and transformants 39, 19 and 30 produced a wPl - l /wPl -4 amplicon. Transformants 21, 34 and 23 did not produce a wPl -l /wPl-4 amplicon, indicating that they were unable to produce a wild type opill transcript. - 125 -Kb L WT 39 19 21 34 30 23 1. 1.0—> 0.5—> Figure 4-7: PCR with the primer pair wPl - l / rP l -2 on the genomic D N A of 6 selected transformants and wild type 0. piliferum. 200 ng of genomic D N A of each of the transformants and the wild type (WT) was amplified using the w P l - l / r P l - 2 primer pair in PCR reactions using a hybridisation temperature of 56 C with 30 cycles of amplification. L 1 Kb D N A ladder (Life Technologies). - 1 2 6 -L WT 39 19 21 3 4 30 2 3 -ve w p 1 - 1 / w p 1 - 4 N L 1 / N L 2 ^ Figure 4-8: Multiplex RT-PCR using opill specific and ITS specific primer pairs with the cDNA of 6 selected transformants and wild type 0. piliferum. RT reactions were performed as described in section 4.2.7, and the resulting cDNA from each of the transformants and the wild type (WT) was amplified using the NL1/NL2 and wPl - l /wP l -4 primer pairs in multiplex PCR reactions using a hybridisation temperature of 56 C with 40 cycles of amplification. L: 1 Kb D N A ladder (Life Technologies), -ve: No RT wild type control. - 127 -4.3.5. Protease Assays The six transformants and the wild type strain were grown in BSA media for 8 days to determine if their extracellular proteolytic activities, or their growth on protein as a sole nitrogen and carbon sources, was affected. The wild type and transformant 39 had similar growth in the BSA medium, while transformant 19 produced ~30 % less biomass (Figure 4-9). Al l of the other transformants grew substantially less. Transformant 30 produced ~60% less biomass than the wild type, while transformants 21, 34 and 23 produced ~80% less biomass than the wild type. In BSA media supplemented with glucose and ammonia all the transformants and the wild type produced a similar amount of biomass. In BSA media the wild type and transformant 39 produced similar levels of extracellular proteolytic activity, while transformant 19 produced ~20% greater activity than the wild type. Al l other transformants produced highly reduced levels of proteolytic activity. Transformants 34 and 23 produced ~60% less activity than the wild type and transformants 30 and 21 produced ~80% less activity than the wild type. -128 -A Strain g 0.0006 o.ooos -1 WT 39 1 9 21 34 30 23 Strain Figure 4-9: Growth and proteolytic activities of wild type O. piliferum and 6 selected transformants. A) 107 yeast and/or spore cells of transformants 39, 19, 21, 34, 30 and 21 and the wild type (WT) were inoculated into BSA or BSA supplemented with glucose and ammonia and incubated for 8 days. The growth of the cultures was measured as described in section 4.2.1. B) 107 yeast and/or spore cells of transformants 39, 19, 21, 34, 30 and 21 and the wild type were inoculated into BSA and grown for 8 days. The proteolytic activity of the cultures was then measured as described in section 4.2.8. - 129 -4.3.6. Growth on wood To assess the growth of the six transformants and the wild type strain on wood they were inoculated onto pine wood blocks and grown for two weeks (Figure 4-10). Isolates 19, 30 and 39 grew similarly to the wild type. On the other hand, isolates 21, 34 and 23 grew slowly and caused little staining on the wood blocks. Ergosterol analysis of the wood blocks inoculated with transformants 34 and 23, the two isolates with a specifically disrupted opill locus, produced ~50 % less and ~40 % less fungal biomass, respectively, than the wild type (Figure 4-11). -130 -23 Figure 4-10: Wild type O. piliferum (WT) and 6 selected transformants grown on lodge pole pine blocks for two weeks. Transformants 39 (Norm + group), 19 (Over group) , 21 (White group), 34 (Norm - group), 30 (White group), and 21 (Norm — group) were inoculated onto lodge pole pine blocks as fungal plugs and their growth rates and morphologies were compared to the wild type strain after two weeks of growth at 25 C. -131 -16 14 A 12 10 A 8 4 A Control WT 34 23 Figure 4-11: Fungal biomass as determined by ergosterol analysis of wood infected with wild type O. piliferum and selected transformants. The wild type strain (WT) and transformants 34 and 23 were inoculated onto lodgepole pine wood blocks as described in section 4.2.1 and grown for two weeks. The control sample is uninfected wood. Ergosterol was extracted from the wood samples and analysed by GC as described in section 4.2.2. - 132 -4.4. Discussion A PEG-mediated transformation system has been used to transform 0. piceae with an efficiency of ~104 transformants per pg of vector [150]. This method requires the production of protoplasts, which can be difficult and time consuming to prepare, and produces transformants with multiple integrations. ATMT has been reported as an efficient method for transforming filamentous fungi and produced transformants with mainly a single copy of the integrating vector [152] [158]. Using ATMT, transformation efficiencies of 15 and 50 transformants per 107 cells were obtained for O. piceae and 0. piliferum, respectively. Similar transformation efficiencies using ATMT have been reported with many fungi, mcluding M. grammicola, C. immitis, and Fusarium veneatum [152] [156] [157]. The efficiency obtained can vary gready and has been reported to be as low as 1 transformant per 5x107 cells {or A. bisporus to as high as ~5,000 transformants per 107 cells for AT. crassa, A. awamori and F. oxysporum [157] [153]. Transformation efficiencies can vary significandy even between species of the same genus. For example, F. oxysporum and F. circinatum, both had efficiencies of ~5,000 transformants per 107 cells, but F. venenatum had an efficiency of only 25 transformants per 107 cells [153] [154] [157]. This was also reported with different Aspergillus species and A. awamori had an efficiency of -1,000 transformants per 107 cells, while A. niger only produced 5 transformants per 107 cells [157]. -133 -Using ATMT ectopic integrations in S. cerevisiae and M. graminicola occur by iUegitirnate recombination through the T-DNA borders [166] [152]. In this work transformants 39, 19, 21 and 30 contained ectopically integrated copies of the disruption cassette. The random integration of the disruption vector in these transformants likely occurred by a similar mechanism to those described for S. cerevisiae and M. graminicola. Three different types of ectopic transformants were obtained, perhaps indicating the preference of the T-DNA to integrate into specific sites. One type of ectopic transformant showed no morphological alterations, no change in their clearing zone on SMA and grew as well as the wild type on wood. The integration of the disruption vector in these transformants had no apparent effect in any of the experiments performed. From this group, transformant 39 was further characterised and contained only ectopically integrated copies of the disruption cassette. Other ectopic transformants produced an increased clearing zone, grew slowly on M E A and were darker in appearance than the wild type. Al l but one of these transformants grew as well as the wild type on wood. Transformant 19 from this group was further characterised and contained only ectopically integrated disruption vector. This transformant produced ~20% greater extracellular proteolytic activity than the wild type when grown in BSA media, indicating that the disruption cassette may have integrated into a gene involved in protease regulation. This, however, was not confirmed. -134-The last group of ectopic transformants had reduced skim milk clearing ability and were albino on SMA but normal in appearance on M E A . Two of these transformants grew poorly on wood, including transformant 21. This transformant grew at a significandy reduced rate on BSA and produced ~80% less proteolytic activity than the wild type. Transformant 21 contained both ectopically integrated disruption vector, and a copy that had integrated into the opill locus. Transformant 30, from this same group also grew poorly on BSA and produced ~80% less proteolytic activity than the wild type, but seemed to grow normally on wood. This transformant contained only ectopically integrated disruption vector. In these transformants the disruption vector must have randomly integrated into a gene involved in the regulation of protease genes and melanin biosynthesis genes. Both of which are known to be regulated by carbon and nitrogen sources [43]. As the growth of transformant 30 on wood was unaffected, it is unlikely that this gene was important for growth on wood. Transformant 21 contained two copies of the disruption vector. In A. bisporus 2 of 4 transformants contained multiple insertions, in F. circinatum 1 of 8 transformants contained two insertion sites, and in C. immitis two of 21 transformants contained more than one copy of the disruption vector [157] [154] [156]. In M. graminicola none of the 16 transformants screened, contained multiple integrations [152]. These studies show that although integration at a single site is common using ATMT, multiple integrations can occur. ATMT has been reported to increase the frequency of homologous recombination over ectopic integration and ATMT has been successfully used to disrupt genes when other transformation systems had failed [153] [160] [152]. In random integration experiments in - 135 -O. piceae, ~1% of the transformants produced a reduced clearing zone on SMA plates [158]. In this work, ~17% of the 0. piliferum transformants had diminished extracellular proteolytic activity indicating that protease genes were targeted. Of the transformants with diminished extracellular proteolytic activity, five showed no morphological differences with the wild type, including transformants 34 and 23 that were further characterised. Both grew poorly in BSA media, produced ~60% less proteolytic activity than the wild type and produced ~50 % less biomass when grown on wood. In the southern analysis, it was clear that both of these transformants contained a single copy of the disruption vector integrated into the opill locus. Multiplex PCR and PCR analyses indicated that, during integration of the disruption cassette into opill, a portion of the 3' end of the gene was lost. It is not uncommon for deletions to occur during integration events. Similar experiments with C. resinifera performed in our lab also showed that deletions occurred during targeted integration [167] [48]. Multiplex RT-PCR showed that a wild type opill transcript was not being synthesised by these transformants. These results strongly indicated that opill was disrupted and that an Opill subtilase could not have been produced in transformants 34 and 23. No genes have previously been disrupted in any sapstaining species of Ophiosotoma although ~2,000 transformants had been screened in one study [168]. A few genes have been disrupted in the closely related fungus 0. novo-ulmi. The chitin synthetase A gene from this organism was disrupted with a hygromycin phosphotransferase (HPH) gene under the control of an A. nidulans gt>dA promoter [169] [170]. Only one disruptant was isolated and was morphologically indistinguishable from the parent strain. This was likely due to the - 1 3 6 -presence of other chitin synthases with sirnilar functions. A class I a-l,2-mannosidase IA gene was also disrupted in 0. novo-ulmi [171]. One disruptant was isolated and exhibited a "fluffy" morphology and had reduced radial growth. As well, an 0. novo-ulmi mutant unable to produce cerato-ulmi was generated by transformation-mediated gene disruption [172]. In 0. novo-ulmi, all the targeted gene disruptions failed to produce a significandy altered phenotype [169] [171] [172]. In each case, the authors suggested that the presence of redundant or equivalent genes masked the effects of the disruption. Targeted disruption of an alkaline protease gene in A. fumigatus also had no effect, and the authors suggested this was due to the induction of compensatory protease genes [102]. Targeted disruption may also have no effect when the disrupted gene is non-essential, as was reported forprtA, which encodes a serine protease, in A. nidulans [135]. The disruption of opill severely impaired both the level of extracellular proteolytic activity produced and the ability of the O. piliferum to grow on wood and in BSA. Although the biomass produced by the opill disruptants was reduced by ~80 % in BSA media, the biomass produced was only reduced by ~50 % in wood. This is indicative of the complex nature of the wood microenvironment, which contains several nitrogen and carbon sources besides protein. As well, the wood microenvironment may have induced other proteases not expressed during growth in the BSA media; however, expression of these proteases was insufficient to allow normal growth of the disruptants on wood and Opill production was necessary for normal growth to occur. The restoration of the growth of the targeted disruptants in BSA media, supplemented with glucose and ammonia, indicated that it was - 137 -solely the loss of the ability of these mutants to acquire carbon and nitrogen from exogenous protein caused their impaired growth in BSA media and on wood. - 1 3 8 -4.5. Conclusions This is the first targeted gene disruption in any species of Ophiostoma to produce a clearly altered phenotype, and the first successful targeted gene disruption in any sapstaining species of Ophiostoma. In 0. piliferum, ATMT was a suitable method for performing targeted gene disruptions. Genes with functions that are redundant to opill must not exist in 0. piliferum. It is clear that in the microenvironment found in wood, Opill is important in allowing fungal growth and that insufficient levels of other extracellular proteases were produced to allow normal growth of the disruptants. It is also clear that at neutral pH's Opill is important in allowing growth in BSA media. It is highly unlikely that identical or equivalent uncharacterised secondary mutations, leading to the phenotypes found, occurred in the genomes of these transformants. The role of opill in the physiology of 0. piliferum is clearly in the acquisition of nitrogen and carbon from exogenous protein sources. - 1 3 9 -Chapter 5 : Conc lus ions and Future work The overall objective of this thesis was to determine the physiological roles of subtilases, a type of serine protease, in sapstaining fungi. To achieve this objective several steps were taken. First the sequence variability and distribution of subtilase genes in commercially relevant sapstaining fungi was assessed. Second, the regulation of three representative subtilase genes by exogenous nutrient sources and ambient pH was studied. Third, the targeted disruption of two of these genes was attempted. This work increases our understanding of the biological relationship between sapstaining fungi and wood, which is important as our current understanding of the biology of sapstain is not sufficient to allow the development of new control strategies. Sapstain fungi are a major problem to the forest products industry and millions of dollars are spent every year to prevent the growth of these fungi in sawn wood. Modern treatments are, however, unsatisfactory. To develop new control strategies it is essential to have a better understanding of how sapstain fungi grow in wood. One of the least understood aspects of this process is how sapstain fungi are able to acquire nitrogen. As proteins are the major form of nitrogen found in trees the production of proteases by sapstain fungi are likely critical to their growth in wood. However, to date littie work has been done on the proteolytic systems of these fungi. The sapstaining fungus 0. floccosum 387N has been shown to produce a subtilase as its dominant extracellular protease. This enzyme was subsequendy purified and shown to be able to break down wood proteins. Subtilases have been reported to play a role in the pathogenicity and nutrient acquisition of several fungi. - 140 -However, no information was available on the ability of sapstaining species aside from O. floccosum 387N to produce these enzymes. The work presented in this thesis shows that subtilase genes are ubiquitous in sapstain fungi. Partial sequence data from fourteen subtilase genes and the full-length sequences of seven subtilases were obtained from different sapstain species. This almost doubles the number of available fungal subtilase sequences. The possible segregation of intracellular and extracellular subtilases by phylogenetic analysis had been reported. This work confirms this and further segregates extracellular subtilases into two groups. Using Southern blot analyses the copy number and distribution of subtilase genes, in representative sapstain species, was determined. It was determined that most Ophiostoma species contain Ofloc 1 group homologs, while homologs from the Opic group are only present in certain Ophiostoma species. Homologs from these two groups were not found in either C. resinifera or Leptographium sp., possibly indicating that these species do not produce extracellular subtilases. Cr group genes were identified in all of the species tested, except O.piceae. Little was known about how proteolytic systems are regulated in sapstaining fungi and previous research only looked at extracellular proteolytic activities. This provided no information on the regulation of specific genes, fn this thesis, the regulation of three subtilase genes opi/1, opic and opil2 was assessed in response to available nutrients, pH and culture age. This work showed that opill is induced by BSA but is not affected by either nitrogen or carbon repression. Induction of this gene occurred witiiin twelve hours of the inoculation of the cultures into BSA media. pH is able to override this induction and opill expression was not found at acidic pH's. opic expression is also induced by exogenous - 141 -protein but requires the removal of either nitrogen or carbon repression. The induction of this gene was shown to occur within 24 hours of growth in BSA media. The expression of opic is also repressed in acidic conditions, again mdicating the ability of pH regulatory pathways to override induction signals, opi/2 was expressed under all of the conditions tested. These data suggest that Opill and Opic are involved in nutrient acquisition and that Opil2 likely plays a housekeeping role. A purification scheme has been developed for purifying a subtilase from 0. floccosum 387N but this technique was slow and inefficient. To perform biochemical analyses on Opil l , Opic and Opil2 large quantities of these enzymes were required. A system using E. coli as a host for the heterologous expression of these enzymes as his-tagged fusion proteins was therefore developed to allow their easy purification by affinity chromatography. Although, the purified proteins had no detectable proteolytic activities they may be useful for antibody development and cellular localisation studies. Other researchers have suggested the possible roles of several fungal subtilases but little had been done to prove these roles. This thesis used gene disruption in an attempt to prove that Opill and Opic are involved in nutrient acquisition during fungal growth in wood. An A. tumefaciens mediated transformation system was first developed to perform the gene disruptions in 0. piceae and 0. piliferum. Unlike the previously developed P E G mediated transformation system for 0. piceae this system produced a high number of transformants containing a single copy of the disruption vector. Although, no 0. piceae mutants were obtained with a disrupted opic gene, several protease deficient mutants were obtained from the transformation of 0. piliferum. Several of these transformants contained random - 142-integrations of the disruption vector but two disruptants had the hygromycin expression cassette inserted solely into their opill loci. These two mutants had gready reduced growth in BSA and significandy lower extracellular proteolytic activity. Both mutants also had greatly reduced growth in wood, indicating that Opill is involved in the acquisition of nutrients from wood. This is the first reported gene disruption in any sapstaining species of Ophiostoma, and the first report to give clear evidence of the physiological role of a fungal subtilase. This thesis extends what is known about subtilases in general. The work presented could provide useful information to other researchers working on fungal subtilases, providing them with clues to the possible roles the subtilases they are studying may play. Also, this work shows the importance of subtilases in the growth of sapstain fungi in wood, providing us with new information about the biology of sapstain. This could lead to the development of new control strategies. Although it is likely that other members of the Oflocl group share a similar role to Opil l , this needs to be proven. The role of Opic was not resolved and it would be useful in future work to reattempt the disruption of this gene. To do this it would be advisable to use longer homologous flanking sequences than those used in this study. Longer regions of homolgy should increase the chances of homologous recombination. As well, a counterselectable marker could be employed to aid in finding disruptants. In this technique, a second marker is added to the disruption cassette outside the regions of homology. Targeted disruptants can then be identified by the loss of this second marker. The role of Opil2 was also not confirmed. Although it seems clear that this enzyme is intracellular, its' exact role is - 143 -uncertain. Future work could also attempt to clarify the role of this gene by disrupting it. Although, a heterologous expression system for Opil l , Opil2, and Opic was developed the purified enzymes were inactive. It would also be useful to express these subtilases, perhaps using S. cerevisiase as a host, with intact pre-pro regions to obtain fully active proteases. These enzymes could then be used to determine their substrate specificities and other biochemical properties. The role that other types of extracellular proteases play in sapstain is not known. Several fungi produce aspartyl proteases as their dominant extracellular protease. As no extracellular subtilase genes were found in either C. resinifera of Leptographium sp. perhaps these species use aspartyl proteases as their dominant extracellular protease. Future work on aspartyl and other proteases in these fungi might provide important clues about why the physiology of Ceratocystis spp. and Ophiostoma spp. are so different, with Ceratocystis spp. growing mainly in logs and freshly cut lumber and Ophiostoma spp. growing primarily in sawn wood. - 144 -References 1. COFI. 2000. COFI BC Fact Book., Council of Forest Industries. 2. Seifert, K. A. 1993. Sapstain of commercial lumber by species of Ophiostoma and Ceratocystis, The American Phytopathological Society, St-Paul, M N . 3. Seifert, K . A., Breuil, C , Rossignol, L., Best, M. , Saddler, J. N . 1988. Screening for microorganisms with the potential for biological control of sapstain on unseasoned lumber. Material und Organismen 23: 81-95. 4. Behrendt, C. J., Blanchette, R. A., Farrell, R. L. 1995. Biological control of blue-stain fungi in wood. Phytopathology 85: 92-96. 5. Yang, D. Q., Rossignol, L. 1999. Evaluation of Gliocladium roseum against wood-degrading fungi in vitro and on major Canadian wood species. Biocontrol Sci Tech 9: 409-420. 6. Behrendt, C. J., Blanchette, R. A., Farrell, R. L. 1995. An integrated approach, using biological and chemical control, to prevent blue stain in pine logs. Can J Bot 74: 613-619. 7. Whitemcdougall, W. J., Blanchette, R. A., Farrell, R. L. 1998. Biological control of blue stain fungi on Populus tremuloides using selected Ophiostoma isolates. Holzforschung 52: 234-240. 8. Dawson-Andoh, B. E., Morrell, J. J. 1992. Extraction of proteins from wood wafers colonized by decay fungi. Holzforschung 46: 117-120. 9. Uzunovic, A., Webber, J. F., Peace, A. J., Dickinson, D. J. 1999. The role of mechanized harvesting in the development of bluestain in pine. Can J Forest Res 29: 242-251. 10. Kaarik, A. 1980. Fungi causing sapstain in wood, The Swedish University of Agricultural Sciences, Uppsala, Sweden. 11. Uzunovic, A., Yang, D. Q., Gagne, P., Breuil, C , Bernier, L., Byrne, A., Gignac, M. , Kim, S. H . 1999. Fungi that cause sapstain in Canadian softwoods. Can J Microbiol 45: 914-922. - 145 -12. Harrington, T. C. 1981. Cyclohexirnide sensitivity as a taxonomic character in Ceratocystis. Mycologia 73: 1123-1129. 13. Weijman, A. C. M. , De Hoog, G. S. 1975. On the Subdivision of the Genus Ceratocystis. Antonie Van Leeuwenhoek J Microbiol Serol 41: 353-360. 14. Hausner, G., Reid, J., Klassen, G. R. 1993. On the subdivision of Ceratocystis s.L, based on partial ribosomal D N A sequences. Can J Bot 71: 52-63. 15. Spatafora, J. W., Blackwell, M. 1994. The polyphyletic origins of Ophiostomatoid fungi. MycolRes 98: 1-9. 16. Hausner, G., Reid, J., Klassen, G. R. 2000. On the phylogeny of members of Ceratocystis s.s. and Ophiostoma that possess different anamorphic states, with emphasis on the anamorph genus Leptographium, based on partial ribosomal D N A sequences. Can J Bot 78: 903-916. 17. Kim, S., Han, A., Kronstad, J., Breuil, C. 1999. Differentiation of sapstain fungi by restriction fragment length polymorphism patterns in nuclear small subunit ribosomal D N A . FEMS Micro Lett 177: 151 - 157. 18. Pipe, N . D., Buck, K. W., Brasier, C. M . 1995. Genomic fingerprmting supports the separation of Ophiostoma piceae into two species. Mycol Res 99: 1182-1186. 19. Pipe, N . D., Brasier, C. M. , Buck, K. W. 2000. Evolutionary relationships of the Dutch elm disease fungus Ophiostoma novo-ulmi to other Ophiostoma species investigated by restriction fragment length polymorphism analysis of the rDNA region. J Phytopathology 148: 533-539. 20. Kim, S. H. , Breuil, C. 2001. Common nuclear ribosomal internal transcribed spacer sequences occur in the sibling species Ophiostoma piceae and O. quercus. Mycol Res 105: 331-333. 21. Harrington, T. C , McNew, D., Steimel, J., Hofstra, D., Farrell, R. 2001. Phylogeny and taxonomy of the Ophiostoma piceae complex and the Dutch elm disease fungi. Mycologia 93: 111-136. 22. Schroeder, S., Kim, S. H., Cheung, W. T., Sterflinger, K., Breuil, C. 2001. Phylogenetic relationship of Ophiostoma piliferum to other sapstain fungi based on the nuclear rRNA gene. FEMS Microbiol Lett 195: 163-167. 23. Witthuhn, R. C , Wingfield, B. D., Wingfield, M . J., Harrington, T. C. 1999. PCR-based identification and phylogeny of species of Ceratocystis sensu stricto. Mycol Res 103: 743-749. -146-24. Loppnau, P., Breuil, C. 2003. Species level identification of conifer associated Cerotcystis sapstain fungi by PCR-RFLP on a P-rabulin gene fragment. FEMS Microbiol Lett, accepted. 25. Brasier, C. M. , Kirk, S. A. 1993. Sibling species witibin Ophiostoma piceae. Mycol Res 97: 811-816. 26. Kim, S. PL, Han, A., Uzunovic, A., Breuil, C. 1998. Specificity of the universal ribosomal D N A primers against softwood sapstain fungi. Material und Organismen (Berlin) 32: 183-193. 27. Kim, S. H., Uzunovic, A., Breuil, C. 1999. Rapid detection of Ophiostoma piceae and 0. quercusm stained wood by PCR. App Environ Microbiol 65: 287-290. 28. Tanguay, P., Racine, G., Dufour, J., Bernier, L., Breuil, C. 2003. Intra and interspecific karyotype variations in Ophiostoma species. In preparation. 29. Gagne, P., Yang, D. Q., Hamelin, R. C , Bernier, L. 2001. Genetic variability of Canadian populations of the sapstaining fungus Ophiostoma piceae. Phytopathology 91: 369-376. 30. Bernier, L., Racine, G., St-Michel, E., Kim, S. H., Breuil, C. 2001. Genetic variability of Ophiostoma piliferum in Canada. Phytopathology 91. 31. Jacobs, K., Wingfield, M . J. 2001. Leptographium species: Tree pathogens, insect associates and agents of blue-stain, APS Press, St. Paul, Minnesota. 32. Harrington, T. C , Steimel, J. P., Wingfield, M . J., Kile, G. A. 1996. Isozyme variation and species delimitation in the Ceratocystis coerulescens complex. Mycologia 88: 104-113. 33. Witthuhn, R. C , Harrington Thomas, C , Steimel Joseph, P., Wingfield Brenda, D., Wingfield Michael, J. 2000. Comparison of isozymes, rDNA spacer regions and MAT-2 D N A sequences as phylogenetic characters in the analysis of the Ceratocystis coerulescens complex. Mycologia 92: 447-452. 34. Malloch, D., Blackwell, M . 1993. Dispersal biology of the Ophiostomatoid fungi, American Phytopathological Society, St. Paul, USA. 35. Klepzig, K . 1998. Competition between a biological control fungus, Ophiostoma piliferum, and symbionts of the southern pine beede. Mycologia 90: 69-75. - 147 -36. Krokene, P., Solheim, H . 1997. Growth of four bark-beetle-associated blue-stain fungi in relation to the induced wound responses in Norway spruce. Can J Bot 75: 618-625. 37. Harrington, T. C. 1993. Diseases of conifers caused by species of Ophiostoma and Leptographium, American Phytopathological Society, St. Paul, USA. 38. Harrington, T. C , Wingfield, M. J. 1998. The Ceratocystis species on conifers. Can J Bot 76: 1446-1457. 39. Kaarik, A. 1960. Growth and sporulation of Ophiostoma and some other blueing fungi on synthetic media. A-B Symb Bot Upsal 16: 1-168. 40. Lagerberg, T., Lundberg, G., Melin, E. 1927. Biological and practical researches into blueing in pine and spruce. Skogsvardsforeningens Tidskrift 10: 147-272, 561-695. 41. Seifert, K. A., Grylls, B. T. 1993. A survey of the sapstaining fungi of Canada, Forintek Canada Corporation, Vancouver, B.C. Canada. 42. Money, N . P. 1997. Wishful thinking of turgor revisited: The mechanics of fungal growth. Fung Gen Biol 21: 173-187. 43. Eagen, R., Breuil, C. 1997. The sap-stairiing fungus Ophiostoma piceae synthesizes different types of melanin in different growth media. Can J Microbiol 43: 592-595. 44. Fleet, C , Breuil, C. 2002. Inhibitors and genetic analysis of scytalone dehydratase confirm the presence of DHN-melanin pathway in sapstain fungi. Mycol Res 106: 1331-1339. 45. Wang, H. L., Breuil, C. 2002. A second reductase gene involved in melanin biosynthesis in the sap-staining fungus Ophiostoma floccosum. Mol Genet Genom 267: 557-563. 46. Eagen, R., Kim, S. H., Kronstad, J. W., Breuil, C. 2001. A hydroxynaphthalene reductase gene from the wood-staining fungus Ophiostoma floccosum complements the buff phenotype in Magnaporthegrisea. Mycol Res 105: 461-469. 47. Wang, H . L., Kim, S. H., Breuil, C. 2001. A scytalone dehydratase gene from Ophiostoma floccosum restores the melanization and pathogenicity phenotypes of a melanin-deficient Colletotrichum lagenarium mutant. Mol Genet Genom 266: 126-132. 48. Loppnau, P. 2003. Canadian Populations and Melanin Biosynthetic Genes of Ceratocystis resinefera. M.Sc. Thesis. University of British Columbia, Vancouver, B.C. - 1 4 8 -49. Fleet, C. 2001. Growth, nutrition and genetic factors that affect pigmentation of wood-sapstain fungi. M.Sc. Thesis. University of British Columbia, Vancouver, Canada. 50. Lindahl, B., Finlay, R., Olsson, S. 2001. Simultaneous, bi-directional translocation of 32P and 33P between wood blocks connected by mycelial cords of Hjpholoma fascicular*. New Phytologist 150: 189-194. 51. Rayner, A. D. M. , Boddy, L. 1988. Fungal decomposition of wood, John Wiley & Sons. 52. Gibbs, J. N . 1993. The biology of Ophiostomatoid fungi causing sapstain in trees and freshly cut logs, American Phytopathological Society, St. Paul, USA. 53. Zink, P., Fengel, D. 1988. Studies on the colouring matter of blue-stain fungi. Part 1. General characterisation and the associated compounds. Holzforschung 42: 217-220. 54. Nilsson, T. 1973. Studies on wood degradation and cellulolytic activity of microfungi. Studia Forestalia Suecica 104: 2-40. 55. Sithole, B. B., Sullivan, J. L., Allen, L. H . 1992. Identification and quantitation of acetone extractives of wood and bark by ion exchange and capillary GC with a spreadsheet program. Holzforschung 46: 409-416. 56. Fengel, D., Wegner, G. 1984. Wood chemistry, ultrastructure, reactions, Walter de Gruyter, Berlin. 57. Gao, Y., Breuil, C. 1995. Extracellular lipase production by a sapwood-staining fungus, Ophiostoma piceae. World J Microbiol Biotech 11: 638-642. 58. Abraham, L., Hoffman, B., Gao, Y., Breuil, C. 1998. Action of Ophiostoma piceae protease and lipase on wood nutrients. Can J Microbiol 44: 698-701. 59. Gao, Y., Chen, T., Breuil, C. 1995. Identification and quantification of non-volatile Hpophilic substances in fresh sapwood and heartwood of lodgepole pine {Pinus contorta Dougl.). Holzforschung 49: 20-28. 60. Blanchette, R. A. 1992. Anatomical responses of xylem to injury and invasion by fungi, Springer-Verlag, Berlin, Heidelberg, New York, 76-95 pp. 61. Brush, T., Farrell, R. L., Ho, C. 1994. Biodegradation of wood extractives from southern yellow pine by Ophiostoma piliferum. TAPPI Journal 7 7 : 155-159. - 149 -62. Chen, T., Breuil, C , Carriere, S., Hatton, J. 1994. Solid-phase extraction can rapidly separate lipid classes from acetone extracts of wood and pulp. TAPPI Journal 77: 236-240. 63. Fischer, C , H611, W. 1992. Food reserves of Scots pine (Pinus sy/vestrisL,.), II. Seasonal changes and radial distribution of carbohydrate and fat reserves in pine wood. Trees 6: 147-155. 64. Saranpaa, P., Holl, W. 1989. Soluble carbohydrates of Pinus sylvestris L. sapwood and heartwood. Trees 3: 138-143. 65. Merrill, W., Cowling, E. B. 1966. Role of nitrogen in wood deterioration. Amounts and distribution of nitrogen in tree stems. Can J Bot 44: 1555-1580. 66. Kramer, P. J., Kazlowski, T. T. 1979. Physiology of Wood Plants, Academic Press, San Francisco. 67. Sauter, J. J., van Cleve, B., Wellenkamp, S. 1990. Ultrastructural and biochemical results on the localisation and distribution of storage proteins in a poplar and in twigs of other tree species. Holzforschung 43: 1-6. 68. Hao, B.-Z., Wu, J.-L. 1993. Vacuole proteins in parenchyma cells of secondary phloem and xylem of Dalbergia odorifera. Trees 8: 104-109. 69. Sauter, J., van Cleve, B. 1994. Storage, mobilization and interrelations of starch, sugars, protein and fat in the ray storage tissue of poplar trees. Trees 8: 297-304. 70. Abraham, L. D., Breuil, C. 1996. Isolation and characterisation of a subtilisin-like serine protease secreted by the sap-staining fungus Ophiostoma piceae. Enzyme & Microbial Tech 18: 133-140. 71. Sophianopoulou, V., Diallinas, G. 1995. Amino acid transporters of lower eukaryotes: Regulation, structure and topogenesis. FEMS Micro Rev 16: 53-75. 72. Chalot, M. , Brun, A. 1998. Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas. FEMS Micro Rev 22: 21-44. 73. Magasanik, B., Kaiser, C. A. 2002. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290: 1-18. 74. Marzluf, G. A. 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev 61 : 17-32. - 150 -75. Katz, M . E., Hynes, M . 1989. Isolation and Analysis of the acetate regulatory gene, facB, from Aspergillus nidulans. Mol Cell Biol 9: 5696-5701. 76. Burger, G., Strauss, J., Scazzocchio, C , Lang, B. 1991. nirA, the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans, encodes a putative GAL4-type zinc finger protein and contains introns in highly conserved regions. Mol Cell Biol 11: 795-802. 77. Siddiqui, A. H. , Brandriss, M . C. 1989. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences. Mol Cell Biol 9: 4706-4712. 78. Suarez, T., de Queiroz, M . V., Oestreicher, N . , Scazzocchio, C. 1995. The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae. Embo J 14: 1453-1467. 79. Yuan, G. F., Fu, Y. H. , Marzluf, G. A. 1991. nit A, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-bmding domain. Mol Cell Biol 11: 5735-5745. 80. Ruijter, G. J. G., Visser, J. 1997. Carbon repression in Aspergilli. FEMS Microbiol Lett 151: 103-114. 81. Gancedo, J. M . 1998. Yeast carbon catabolite repression. Microbiol Mol Biol Rev 62: 334-361. 82. Ebbole, D. J. 1998. Carbon catabolite repression of gene expression and conidiation in Neurospora crassa. Fung Genet Biol 25: 15-21. 83. North, M. 1982. Comparative biochemistry of the proteases of eucaryotic microorganisms. Microbiol Rev 8: 308-340. 84. Gottesman, S., Maurizi, M. R. 1992. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev 56: 592-621. 85. Deshpande, M . V. 1992. Proteases in fungal morphogenesis. World J Microbiol Biotech 8: 242-250. 86. Santamaria, F., Reyers, F. 1988. Proteases produced during autolysis of filamentous fungi. Trans Brit Mycol Soc 91: 217-220. 87. Warshel, A., Naray-Szabo, G., Sussman, F., Hwang, J. K. 1989. How do serine proteases really work? Biochemistry 28: 3629-3637. - 151 -88. Siezen, R. J., de Vos, W. M., Leunissen, J. A., Dijkstra, B. W. 1991. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteases. Protein Eng 4: 719-737. 89. Siezen, R. J., Leunissen, J. A. 1997. Subtilases: the superfamily of subtihsin-like serine proteases. Protein Sci 6: 501-523. 90. Segers, R., Butt, T., Carder, J. H. , Keen, J. N . , Kerry, B. R., Peberdy, J. F. 1999. The subtilisins of fungal pathogens of insects, nematodes and plants: Distribution and variation. Mycol Res 103: 395-402. 91. Betzel, C , Pal, G. P., Saenger, W. 1988. Three-dimensional structure of protease K at 0.15-nm resolution. Eur J Biochem 178: 155-171. 92. Kolvenbach, C. G., Narhi, L. O., Lazenby, K., Samal, B., Arakawa, T. 1990. Comparative study on protease R, T, and K from Tritirachiam album limber. Int J Pept Protein Res 36: 387-391. 93. Keniry, C. A., L i , D., Ashby, A. M . 2002. Cloning and expression studies during vegetative growth and sexual development of psp2, a serine protease gene from Pjrenope^a brassicae. Biochim Biophys Acta 1577: 159-163. 94. Zhu, X . L., Ohta, Y., Jordan, F., Inouye, M. 1989. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 339: 483-484. 95. Silen, J. L., Frank, D., Fujishige, A., Bone, R., Agard, D. A. 1989. Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J Bacteriology 171: 1320-1325. 96. Markaryan, A., Lee, J. D., Sirakova, T. D., Kolattukudy, P. E. 1996. Specific inhibition of mature fungal serine proteases and metalloproteases by their propeptides. J Bacteriology 178: 2211-2215. 97. Monod, M. , Hube, B., Hess, D., Sanglard, D. 1998. Differential regulation of SAP8 and SAP9, which encode two new members of the secreted aspartic protease family in Candida albicans. Microbiology 144: 2731-2737. 98. Betzel, C , Pal, G. P., Struck, M., Jany, K. D., Saenger, W. 1986. Active-site geometry of protease K . Crystallographic study of its complex with a dipeptide chloromethyl ketone inhibitor. FEBS Lett 197: 105-110. 99. Frederick, G. D., Rombouts, P., Buxton, F. P. 1993. Cloning and characterisation of pepC, a gene encoding a serine protease from Aspergillus niger. Gene 125: 57-64. - 152 -100. Jarai, G., Kirchherr, D., Buxton, F. P. 1994. Cloning and characterisation of thepepD gene of Aspergillus niger which codes for a subtilisin-like protease. Gene 139: 51-57. 101. Tang, C. M. , Cohen, J., Krausz, T., Van Noorden, S., Holden, D. W. 1993. The alkaline protease of Aspergillus fumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis. Infect Immun 61: 1650-1656. 102. Tang, C. M. , Cohen, J., Holden, D. W. 1992. An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity. Mol Microbiol 6: 1663-1671. 103. Kolattukudy, P. E., Lee, J. D., Rogers, L. M. , Zimmerman, P., Ceselski, S., Fox, B., Stein, B., Copelan, E. A. 1993. Evidence for possible involvement of an elastolytic serine protease in aspergillosis. Infect Immun 61: 2357-2368. 104. Jaton-Ogay, K., Suter, M. , Crameri, R., Falchetto, R., Fatih, A., Monod, M. 1992. Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillusfumigatus. FEMS Microbiol Lett 71: 163-168. 105. Reichard, U. , Cole, G. T., Hill, T. W., Ruchel, R., Monod, M . 2000. Molecular characterisation and influence on fungal development of ALP2, a novel serine protease (torn Aspergillus fumigatus. Int J Med Microbiol 290: 549-558. 106. Katz, M . E., Rice, R. N . , Cheetham, B. F. 1994. Isolation and characterisation of an Aspergillus nidulans gene encoding an alkaline protease. Gene 150: 287-292. 107. Tatsumi, H. , Ogawa, Y., Murakami, S., Ishida, Y. , Murakami, K. , Masaki, A., Kawabe, H. , Arimura, H., Nakano, E., Motai, H . 1989. A full length cDNA clone for the alkaline protease from Aspergillus oryyae: structural analysis and expression in Saccharomyces cerevisiae. MolGenet Genom 219: 33-38. 108. Moralejo, F. J., Cardoza, R. E., Gutierrez, S., Lombrana, M. , Fierro, F., Martin, J. F. 2002. Silencing of the aspergillopepsin B (pepB) gene of Aspergillus awamori by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production. Appl Environ Microbiol 68: 3550-3559. 109. Lugli, E. B., Allen, A. G., Wakefield, A. E. 1997. A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases. Microbiology 143: 2223-2236. 110. Paoletti, M. , Castroviejo, M. , Begueret, J., Clave, C. 2001. Identification and characterisation of a gene encoding a subtilisin-like serine protease induced during the vegetative incompatibility reaction in Podospora anserina. Curr Genet 39: 244-252. - 1 5 3 -111. Sreedhar, L., Kobayashi, D. Y., Bunting, T. E., Hillman, B. I., Belanger, F. C. 1999. Fungal protease expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene 235: 121-129. 112. Segers, R., Butt, T. M. , Keen, J. N . , Kerry, B. R., Peberdy, J. F. 1995. The subtilisins of the invertebrate mycopathogens Verticillium chlamydosporium and Metarhiyium anisopliae are serologically and functionally related. FEMS Microbiol Lett 126: 227-231. 113. Bidochka, M . J., Melzer, M . J. 2000. Genetic polymorphisms in three subtiHsin-like protease isoforms (PrlA, PrlB, and PrlC) from Metarhi^ium strains. Can J Microbiol 46: 1138-1144. 114. Geremia, R. A., Goldman, G. H., Jacobs, D., Ardiles, W., Vila, S. B., Van Montagu, M. , Herrera-Estrella, A. 1993. Molecular characterisation of the protease-encoding gene,prbl, related to mycoparasitism by Trichoderma har^ianum. Mol Microbiol 8: 603-613. 115. Bangyeekhun, E., Cerenius, L., Soderhall, K. 2001. Molecular cloning and characterisation of two serine protease genes from the crayfish plague fungus, Aphanomyces astaci. J Invert Path 77: 206-216. 116. Kingsnorth, C. S., Eastwood, D. C , Burton, K . S. 2001. Cloning and postharvest expression of serine protease transcripts in the cultivated mushroom Agaricus bisporus. Fung Genet Biol 32: 135-144. 117. Isogai, T., Fukagawa, M. , Kojo, H. , Kohaska, M. , Aoki, PL, Imanaka, H . 1991. Cloning and nucleotide sequence of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum. Agric Biol Chem 55: 471-477. 118. Johnston, J. M. , Ramos, E. R., Bilbrey, R., Gathman, A., Lilly, W. 2000. Characterisation of ScPrI, a small serine protease, from mycelia of S' chi^ophyllum commune. Mycol Res 104: 726-731. 119. Abraham, L. D., Breuil, C. 1995. Factors affecting autolysis of a subtiksm-like serine protease secreted by Ophiostoma piceae and identification of the cleavage site. Biochimica et Biophysica Acta 1245: 76-84. 120. Gharibian, S., Hoffert, C , Abraham, L. D., Breuil, C. 1996. Localizing an Ophiostoma piceae protease in sapwood of four tree species using polyclonal antibodies. New Phytologist 133: 673-679. 121. Farley, P. C , Sullivan, P. A. 1998. The Rhi^opus ory^ae secreted aspartic protease gene family: an analysis of gene expression. Microbiology 144: 2355-2366. - 154-122. Paterson, I. C , Charnley, A. K., Cooper, R. M. , Clarkson, J. M . 1994. Specific induction of a cuticle-degrading protease of the insect pathogenic fungus Metarhi-^ium anisopliae. Microbiology 140: 185-189. 123. Paoletti, M. , Clave, C , Begueret, J. 1998. Characterisation of a gene from the filamentous fungus Podospora anserina encoding an aspartyl protease induced upon carbon starvation. Gene 210: 45-52. 124. Denison, S. 2000. pH regulation of gene expression in fungi. World J Micro Biot 8: 242-250. 125. Tibbett, M. , Sanders, F. E., Cairney, J. W. G., Leake, J. R. 1999. Temperature regulation of extracellular proteases in ectomycorrhizal fungi (Hebeloma spp.) grown in axenic culture. Mycol Res 103: 707-714. 126. Mclntyre, M. , Berry, D. R., McNeil, B. 2000. Role of proteases in autolysis of Penicillium chrysogenum chemostat cultures in response to nutrient depletion. App Microbiol Biotech 53: 235-242. 127. Sanglard, D., Hube, B., Monod, M. , Odds, F. C , Gow, N . A. R. 1997. A triple deletion of the secreted aspartyl protease genes SAP4, SAPS, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 8: 3539-3546. 128. Hube, B., Sanglard, D., Odds, F. C , Hess, D., Monod, M. , Schafer, W., Brown, A. J., Gow, N . A. 1997. Disruption of each of the secreted aspartyl protease genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect Immun 65: 3529-3538. 129. De Bernardis, F., Arancia, S., Morelli, L., Hube, B., Sanglard, D., Schafer, W., Cassone, A. 1999. Evidence that members of the secretory aspartyl protease gene family, in particular SAP2, are virulence factors for Candida vaginitis. J Infect Dis 179: 201-208. 130. Hube, B., Naglik, J. 2001. Candida albicans proteases: resolving the mystery of a gene family. Microbiology 147: 1997-2005. 131. Gente, S., Durand-Possereau, N . , Fevre, M . 1997. Controls of the expression of aspA, the aspartyl protease gene from Penicillium roqueforti. Mol Genet Genom 256: 557-565. 132. Gente, S., Billon-Grand, G., Poussereau, N . , Fevre, M . 2001. Ambient alkaline pH prevents maturation but not synthesis of ASPA, the aspartyl protease from Penicillium roqueforti. Mol Genet Genom 256: 557-565. - 155 -133. Poussereau, N . , Gente, S., Rascle, C., Billon-Grand, G., Fevre, M. 2001. aspS encoding an unusual aspartyl protease from Sclerotinia sckrotiorum is expressed during phytopathogenesis. FEMS Microbiol Lett 194: 27-32. 134. Poussereau, N . , Creton, S., Billon-Grand, G., Rascle, C., Fevre, M . 2001. Regulation of acpl', encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sckrotiorum. Microbiology 147: 717-726. 135. vanKuyk, P. A., Cheetham, B. F., Katz, M. E. 2000. Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Fung Genet Biol 29: 201-210. 136. Katz, M . E., Flynn, P. K., vanKuyk, P. A., Cheetham, B. F. 1996. Mutations affecting extracellular protease producation in the filamentous fungus Aspergillus nidulans. Mol Genet Genom 250: 715-724. 137. Ausubel, F. M. , Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. 1994. Current Protocols in Molecular Biology, Greene PubHshing Associates and Wiley-Interscience, New York, NY. 138. Thompson, J. D., Higgins, D. G., Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680. 139. Burge, C , Karlin, S. 1997. Prediction of complete gene structures in human genomic D N A . J Mol Biol 268: 78-94. 140. Fickett, J. W., Hatzigeorgiou, A. G. 1997. Eukaryotic promoter recognition. Genome Research 7: 861-878. 141. Moser, M. , Menz, G., Blaser, K., Cramer, R. 1994. Recombinant expression and antigenic properties of a 32-Kilodalton extracellular alkaline protease, representing a possible virulence factor horn. Aspergillus fumigatus. Infect Immun 3: 936-942. 142. Abraham, L. 1995. Functions of a protease secreted by the sap-staining fungus Ophiostoma piceae. PhD Thesis. University of British Columbia, Vancouver, Canada. 143. Tibbet, M. , Sanders, F. E., Cairney, J. W., Leake, J. R. 1999. Temperature regulation of extracellular proteases in ectomychorrhizal fungi (Hebelomoa spp.) grown in anexic culture. Mycol Res 103: 707-714. 144. Van den Homberg, J. P. T. W., van de Vondervoot, P. J. I., Fraissinet-Tachet, L., Visser, J. 1997. Aspergillus as a host for heterologous protein production: the problem of proteases. TIBTECH 15: 256-263. - 156-145. Goldman, G. H. , Montagu, M. V., Herrera-Estrella, A. 1990. Transformation of Trichoderma hannanum by high-voltage electric pulse. Curr Genet 17: 160-174. 146. Sanchez, O., Aguirre, J. 1997. Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. http://wwwkumcedu/research/fgsc/mainhtml. 147. Hinnen, A., Hicks, J. B., Fink, G. R. 1978. Transformation of yeast. Proc Nad Acad Sci USA 75: 1929-1933. 148. Shorde, D., Haber, J. E., Botstein, D. 1982. Lethal disruption of the yeast actin gene by integrative D N A transformation. Science 217: 371-373. 149. Rothstein, R. 1991. Targeting, disruption, replacement, and allele rescue: integrative D N A transformation in yeast. Methods Enzymol 194: 281-301. 150. Wang, H . L., Kim, S. H., Siu, H., Breuil, C. 1999. Transformation of sapstaining fungi with hygromycin B resistance plasmids pAN7-l and pCB1004. Mycol Res 103: 77-80. 151. Royer, J. C , Dewar, K., Hubbes, M. , Horgen, P. A. 1991. Analysis of a high frequency transformation system for Ophiostoma ulmi the causal agent of Dutch elm disease. Mol Genet Genom 225: 168-176. 152. Zwiers, L. -H. , De Ward, M . A. 2001. Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerellagraminicola. Curr genet 39: 388-393. 153. Mullins, E. D., Chen, X. , Romaine, P., Raina, R., Geiser, D. M . , Kang, S. 2001. Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenisis and gene transfer. Phytopathology 91: 173-180. 154. Covert, S. F., Kapoor, P., Lee, M. H., Briley, A., Nairn, C. J. 2001. Agrobacterium tumefaciens mediated transformation of Fusarium circinatum. Mycol Res 105: 259-264. 155. Mikosch, T. S. P., Lavrijssen, B., Sonnenberg, A. S. M. , van Griensven, L. 2001. Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens. Curr Genet 39: 35-39. 156. Abuodeh, R. O., Orbach, M. J., Mandel, M . A., Das, A., Galgiani, J. N . 2000. Genetic transformation of Coccidiodes immitis facilitated by Agrobacterium tumefaciens. J Infect Dis 181: 2106-2110. - 157-157. deGroot, M. , Bundock, P., Hooykaas, P., Beijersbergen, A. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16: 839 -842. 158. Tanguay, P., Breuil, C. submitted. Transforming the sapstaining fungus Ophiostoma piceae with Agrobacterium tumefaciens. Can J Microbiol. 159. Loppnau, P., Tanguay, P., Breuil, C. submitted. Isolation and disruption of a polyketide synthase (PKS1) from Ceratocystis resinifera involved in Dihydroxynaphthalene melanin biosynthesis. 160. Bundock, P., Mroczek, K., Winkler, A. A., Steensma, H . Y., Hooykaas, P. J. J. 1999. T-DNA from Agrobacterium tmefaciens as an efficient tool for gene targeting in Klyveromyces lactis. Mol Genet Genom 261: 115-121. 161. Pikaard, C. 2002. Agrobacterial transformation, Craig Pikaard's Laboratory at University of Washington. 162. Pasanen, A.-L. , Yli-Pietila, K., Pasanen, P., Kalliokoski, P., Tarhanen, J. 1999. Ergosterol Content in Various Fungal Species Biocontaminated Budding Materials. App Environ Microbiol 6 5 : 138-142. 163. Carroll, A., Sweigard, J., Valent, B. 1994. Improved Vectors for Selecting Resistance to Hygromycin. Fung Genet Newsl 4 1 : 22. 164. Hooykaas, P. J. J., Roobol, C , Schilperoort, R. A. 1979. Regulation of the Transfer of T-I Plasmids oi Agrobacterium tumefaciens.} Gen Microbiol 110: 99-110. 165. Bundock, P., den Dulk-Ras, A., Beijersbergen, A., Hooykaas, P. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. E M B O J 14: 3206-3214. 166. Bundock, P., Hooykaas, P. 1996. Integration oi Agrobacterium tumefaciens'T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Nad Acad Sci USA 9 3 : 15272-15275. 167. Fincham, J. R. S. 1989. Transformation in fungi. Microbiol Rev 5 3 : 148-170. 168. Wang, H . L. 2002. Cloning and Characterisation of Melanin Synthesis Genes in Ophiostoma floccosum 387N. PhD Thesis. University of British Columbia, Vancouver, BC. - 158 -169. Hintz, W. E. 1999. Sequence analysis of the chitin synthase A gene of the Dutch elm pathogen Ophiostoma novo-ulmi indicates a close association with the human pathogen Sporothrix schenckii. Gene 237: 215-221. 170. Punt, P. J., Dingemanse, M . A., Jacobs-Meijsing, B. J., Pouwels, P. H. , van den Hondel, C. A. 1988. Isolation and characterisation of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. Gene 69: 49-57. 171. Eades, C. J. 2002. Characterisation of the class I alpha-mannosidase gene family in filamentous fungi. PhD Thesis. University of Victoria, Vicotria, BC, Canada. 172. Bowden, C. G., Smalley, E., Guries, R. P., Hubbes, M. , Temple, B., Horgen, P. A. 1996. Lack of association between cerato-ulmin production and virulence in Ophiostoma novo-ulmi. Mol Plant-Microbe Interac 9: 556-564. - 159 -Appendix 1: Lipase screening Screening of strains of sapstaining fungi for lipase production The initial goal of this thesis was to determine the physiological roles of lipases in the growth and pigmentation of sapstaining fungi. The following appendix describes the work done towards this goal. This first involved screening strains for their ability to produce lipases. Second a lipase from O.floccossum 387N was purified and the N-terminal sequences of three lipase fragments were obtained. This protein sequence information was then used to design a number of degenerate PCR primers and oligonucleotide probes in attempts to clone a lipase gene by PCR and library screening. As well, an expression library was created and screened by lipase activity assays. No lipase gene was obtained from these analyses and work was eventually begun on the proteases produced by sapstaining fungi. As progress was made with that work, further work on the lipase aspect of the project was eventually stopped. Thirty-four strains of sapstaining fungi were tested in liquid culture and on plates for lipase activity. Liquid cultures were grown in olive oil media and an aliquot of the supernatant was then taken for subsequent lipase assays with p-nitrophenyllaurate; spirit blue agar was used for plates. From the results shown in Table 1, it can be noted that neither the growth on the spirit blue agar nor the colour of the media correlated with the amount of lipase activity in liquid culture. In fact the colour of the media rather showed the pH of the media as the - 1 6 0 -Table 1: Lipase activity analysis of 34 strains of sapstaining fungi on spirit blue agar plates and in olive oil liquid cultures Species Units lipase activity Growth on spirit blue agar'' Colour of spirit blue agar Melanization" 0.42 S 3 N 0.44 S 3 N Ophiostoma piceae 0.40 S 3 N 0.24 S 3 N 0.07 S 3 N 0.39 S 4 N Ophiostoma floccosum 0.33 S 4 N 0.314 S 4 N 0.42 S 4 N 0.22 S 2 Y Ophiostoma piliferum 0.29 S 2 Y 0.17 S 2 N 0.33 S 2 N 0.03 S 0 Y 0.15 M 1 Y Leptographium 0.03 S 0 Y 0.02 S 0 Y 0.08 M 2 Y 0.01 S 1 Y C.coerulescens 0.01 S 1 Y 0.04 S 1 Y 0.38 S 3 N 0. setosum 0.39 S 4 N 0.32 S 3 N 0.43 M 3 N 0. coronatum 0.08 M 3 N 0.09 M 3 N 0.25 S 2 N 0. minus 0.03 s 3 N 0.22 s 2 Y 0.22 s 3 Y 0.02 M 2 Y jA.pullulans 0.03 M 3 Y 0.29 M 3 Y "'indicates units of lipase assay in the lOOul of assayed culture supernatant after the culture was grown for four days ^Relative amount of growth on the spirit blue agar after four days of growth: S-strong, M-medium, W-weak ^Indicates the final color of the spirit blue agar after four days of growth with 0 being white and 5 being dark blue "Indicates whether or not melanization of the mycelia was noted after four days of growth: Y-yes, N-no - 161 -spirit blue agar turned dark blue in acidic conditions and light blue in alkaline conditions. No halo was formed on any of the plates, which occurs with lipase producing yeast and bacteria. The fungi were not growing in colonies with distinct edges, but rather as mycelia, which tended to spread out well past the edge of the bulk of the colony, thereby obscuring any potential halo. The spirit blue agar plates were therefore not a useful way of determiriing lipase activity in fungal strains. The amount of growth in liquid culture was not determined as the cultures grew in a mixture of yeast type cells and mycelia making this type of analysis very difficult. From Table 1 it can be seen that most strains within a species produced similar activities; however, several interesting exceptions should be noted. For instance one of the 0 piceae strains produced an activity of one sixth that of the other strains, as did one of the 0.minus strains. Also, one of the 0. coronatum strains and an A. pullulans strain had activities approximately ten times greater than the other strains. Purification and Protein sequences Lipase was prepared from cultures of 0. floccosum 387N grown in olive oil media and purified using hydrophobic interaction and ion exchange chromatography. It was determined that several enzymes were present in these sample preparations and it was critical to determine which of the proteins had lipolytic activity. Native gels were run and the activity stain Fast Blue RR used to identify bands with esterase like activity. In these experiments it was found that the major band in the preparations, at around 35kDA, was the only band with lipolyitc activity. The next step was to obtain amino acid sequence information from this enzyme. In order to achieve this goal the enzyme needed to be fragmented and the individual fragments sequenced. At first we attempted to do the fragmentation using a-chymotrypsin. - 1 6 2 -The purity of the a-chymotrypsin preparations was poor and when SDS-PAGE gels were run on the fragmented samples contaminating bands thoroughly obscured the gels making it impossible to accurately identify lipase protein fragments. An effort was then made to use a-chymotrypsin fused to silica beads allowing the a-chymotrypsin to simply be spun out of solution before SDS-PAGE. With this technique several possible lipase fragments were generated. We then attempted to sequence a few of these fragments. We were not able to obtain sufficient quantities of any of the fragments to get accurate sequencing results. We decided that it would be easier to send a sample of the lipase to the Harvard Microchemistry for fragmentation and automated sequencing. In order to do this a substantial quantity of very pure lipase needed to be obtained. A scaled up version of the purification scheme developed by Gao [173] was used, and the obtained purified lipase samples run out on native gels. The band, that we had previously identified as having lipolytic activity, was cut out and the contained enzyme electroeluted from the gel. This sample was then sent out for analysis. Three of the major fragments obtained were sequenced (Table 2). Only one of these fragments had significant homology to other known lipase sequences and we were able to tentatively locate it about 20 residues 3' to the active site. Unfortunately due to the lack of conservation in lipases we were not able to locate regions of significant similarity in other fungal lipases with the other two fragments obtained. - 163 -Table 2: Obtained Amino Acid Sequences of Lipase Fragments Fragment Name Amino Acid Sequence Similar Sequences Possible Location GT99 GYTVDVFTYGSP 1 .Humicola Lipase (91%) 2. Fusarium heterospomm Lipase (83%) 3' to active site GT113 T L T D M L G F I G Y D P V K Penicillium camemhertii Lipase (66%) Unknown GT117 I G N E A L V T Y L S T O G P V Beta-xylosidase (69%) Unknown P C R analysis PCR primers were then designed, using both the sequence information mentioned above and regions of homology between other fungal lipases. Primers based on regions of homology were difficult to make for several reasons, the lack of conserved regions in lipases, the lack of known gene sequence information from fungal Upases, and the presence of serine and other highly degenerate amino acid residues in many of the conserved areas, etc. Primers based on the obtained lipase sequence were just as hard to develop for many of the reasons listed above and also because we did not know the relative locations of these primers. See Table 3 for a complete list of the primers and other oligonucleotides designed. - 164-Table 3: List of oligonucleotides utilised Primer Name Origin Sequence (5' - 3') Use OPL1 N-Terminus ACIACIACIGA(CT)AT(CAT)GA(CT)GC PCR OPL2 Active Site homology GCI(GC)CACC(GC)AGI(CG)(TA)(GA)TG(TG)CC PCR OPL3 N-terminus GCITT(TC)TT(TC)ACICA(AG)TGGGC PCR OPL4 Homology 3' to active site (GA)TTICCIACIC(GT)IGG PCR OPL5 Protein fragment GT117 ATIGGIAA(CT)GA(GA)GCICTIGT PCR OPL6 Protein fragment GT113 ACIGG(AG)TC(AG)TAICCIAT(AG)AA PCR OPL7A Protein fragment GT99 TA(CG)GT(AG)AA1AC(AG)TCIACIGT PCR OPL7B Protein fragment GT99 CC(AG)TAIGT(AG)AAIAC(AG)TCIAC PCR OPL8 Reverse of OPL5 ACIAGIGC(CT)TC(GA)TTICCIAT PCR OPL9 Reverse of OPL2 GTIACIGGICA(CT)(TA)CITT(AG)GG PCR OPL10A Protein fragment GT113 CC(AT)AT(AG)AAUCCUAGCAT(GA)TC PCR OPL10B Protein fragment GT113 CCGAT(AT)AAUCCUAGCAT(GA)TC PCR OPL10C Protein fragment GT113 CC(AT)AT(AG)AAICC(TC)AACAT(GA)TC PCR OPL12A Protein fragment GT99 (GA)CCATAIGTAAAIACGTCIAC PCR OPL12B Protein fragment GT99 (GA)CCATATGA(AG)AACAC(AG)TCIAC PCR OPL12C Protein fragment GT99 (GA)CCATATGAAAACACGTCIAC PCR OPS1 OPL1-8 PCR band T C A C A A T C T C G T T A T C A T C PCR and sequencing OPS2 OPL1-8 PCR band G A T G A T A A C GA G A T T G T G A C G A C G A PCR OPS3 OPL1-8 PCR band A T G A C G G C T A C C A T C G C A G PCR OPS4 OPL1-8 PCR band C T C G T T G C C G A T G A C A A G A PCR OPS5 p L H l sequence T C G T A G C C T A G A G C T G A G C T G Sequencing of p L H l OPS7 p L H l sequence G A C G A C G G T A T C G A T A G T G A Sequencing o f p L H l OPP1 Protein fragment GT99 T C G T A G C C T A G A G C T G A G C T G Screening library OPP6 Protein fragment GT113 ACIGG(AG)TC(AG)TAICCIAT(AG)AA Screening library EMBL1 Left arm of X phage G T A G G C G G A T C T G G G T C G A C PCR EMBL4 Right arm of X phage C G A C C C A G A T C T G G G T C G A C PCR EMBL6 Right arm of X phage T C T G T C A T C A C G A T A C T G T G A PCR PDT Poly A mRNA tail T T T T T T T T T T T T ^ ^ PCR and reverse transcription - 165 -The degenerate primers designed were then used in a variety of PCR reactions with genomic D N A as a template. Many different reaction conditions and all suitable primer combinations were tested; however, no specific bands were obtained. Many of the degenerate primers designed on homologous sequences produced very high background, possibly indicating the lack of specificity of these primers to lipases. Most of the primers designed from protein sequence on the other hand produced no bands. If any specific bands were produced it is likely that they were so faint as to not be visible or that they were totally obscured by the background bands produced by many of the primers. It was thought that genomic D N A was not an ideal template. In an effort to eliminate some of the background bands produced in our PCR reactions we decided to use cDNA as a template. 0. floccosum 387N was grown on olive oil cellophane plates, to ensure the production of the lipase transcript and to allow for easy collection of the mycelia. Several growth times were tested and it was determined that growing the cultures past 24hrs did not provide better yields, and samples grown beyond 24hrs were more likely to have poor quality RNA, due to increased production of degradative enzymes. Approximately 200mg of mycelia was collected in 2ml Epindorf collection tubes and ground in 300pl of RNA extraction buffer using a specially modified drill bit. These RNA samples were then processed using a modified version of the plant RNA extraction protocol from Qiagen. The Qiagen kit used produced around 45pg of high quality total RNA per 200mg of mycelia. This total RNA was then used in reverse transcriptase reactions to produce cDNA using M M L V reverse transcriptase. -166 -It was necessary to purify the cDNA produced in this reaction before it could be used in PCR reactions. This was likely due to the inhibitory effects of M M L V on Taq polymerase. PCR reactions using this cDNA failed to produce any specific bands as the change from genomic D N A to cDNA still did not remove enough of the background bands produced. Two step PCR reactions, with the first round using a degenerate primer and a poly T primer and the second round using two nested degenerate primers on the products of the first round, also failed to produce specific products. Using the library phage D N A as a template an apparendy specific amplicon was amplified using the primers OPL4 and EMBL1. A subsequent amplicon was obtained by reamplifying this band with the primers OPL1 and OPL8. This band was sequenced and both primer sites were located within the sequence. This sequence did not show any homology to known lipase sequences. In order to identify this band several specific primers were designed from its sequence and used in PCR reactions with the previously designed degenerate primers. No specific products were obtained from these reactions. This could indicate that the sequence obtained was not a lipase sequence, that the degenerate primers could not work in conjunction with the specific primers, or that any specific bands produced were to faint to be easily detected. It is still not known if this sequence is in fact lipase gene sequence or not, to clarify this it would be useful to use this amplicon as a probe to screen the 0. floccosum 387N library. - 167-Screening of library with OPP-1 probe pool Chemiluminescent techniques, with the degenerate probe OPP1 that was designed according to previously obtained protein sequence, were used to screen an 0. floccosum 387N genomic library. Initially around 20 plates at 2-3,000 pfu per plate were screened. Results were unclear as a great deal of background was produced in the cheimluminescent reactions. In order to improve the detection system an alkaline phosphatase detection system was tested instead of the provided horse radish peroxidase system and gave much clearer results on a consistent basis. The library was rescreened with this system and several potentially positive plaques were obtained. These plaques were picked and used to produce dot blots. From these dot blots only one of the plaque pools still produced a signal. This plaque pool was then used to create several subpool plates at about 100 plaques per plate. These plates were screened and several plaques that produced a signal were picked and used to produce a dot blot. Only one of the phage pools produced a dot blot signal. This phage pool was then amplified in liquid culture and the D N A of this amplified phage extracted. The purified D N A was dot blotted and produced a signal. This D N A was then digested with a variety of restriction enzymes. Cleavage with Hind III produced an acceptable restriction fragment pattern for subcloning, and that a 2kb band hybridised with OPP-1 in southern analysis. This purified restriction fragment was then subcloned into pBluescript. -168 -Dot blot analysis was then used to confirm the presence of the insert in transformants. A plasmid containing the correct insert, p L H l , was then amplified and the insert sequenced. The sequence obtained was shown to have no significant homology to other known lipase sequences. A completely homologous binding site to the probe was not located within the obtained sequence; however, several partially homologous binding sites were identified. This indicated that this insert likely did not contain a lipase gene. Several more plates were screened with OPP1, and around 30 plaques that seemed to give a positive signal were picked. These plaques were used to prepare two identical dot blots which were screened with either the OPP1 probe or the OPP6 probe. This was done in order to eliminate any possible background signals from one of the probes. Of the thirty primary pools only one gave a positive signal that showed on both blots. This pool was replated and screened and two plaques produced possible hybridisation signals; however, the signals were weak and they could not be definitively identified as positives. OPP6 was used to confirm if these plaques were indeed positive and it was determined that this probe did not hybridize with these clones, indicating that no lipase sequence was present. For this work to be successful the use of an optimized radioactive screening technique would be necessary as the chemduminescent system used produced a high amount of background signal which was impossible to differentiate from actual hybridisation signals. Creation and Screening of a Lipase Expression Library - 169-The total RNA of 0. piceae 123-142, was extracted and the mRNA obtained purified. The resulting mRNA was used to produce cDNA which was then cloned into p B A D / T O P O thiofusion vectors. This resulting library was screened for the production of active lipase by plating on rhodamine B containing LB agar plates. No lipase producing colonies were discovered. As well colonies were screened for activity using p-nitrophenyllaurate as a substrate in colourometric assays, again no lipase positive colonies were obtained. The library was then screened using the OPP-1 and OPP-6 probes using radioactive methods; however, high background was again an issue and no positive clones were identified. In future work it would be essential to determine what conditions would give optimal results in the hybridisations with these probes. If this optimisation was carried out a lipase clone should be identified. Reference 173. Gao, Y. , Breuil, C. 1998. Properties and substrate specificities of an extracellular lipase purified from Ophiostoma piceae. World J Microbiol Biotech 14: 421-429. 

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