Please read the abstract in the section 00front of this document / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / Unrestricted

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Ophiostoma floccosum and Ophiostoma piliferum are polymorphic ascomycete fungi found throughout the world. Both species are important economically as they are known to colonise timber and cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species, in particular O. piliferum, are used as biological control agents to prevent sapstaining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Other members of the genus include the plant pathogens O. novo-ulmi and O. clavigerum, known to have a severe effect on forest health and economy around the world. O. floccosum and O. piliferum have been demonstrated in the laboratory to be fermented in large volumes and they are particularly suitable as hosts capable of secreting extracellular recombinant proteins. This research aimed to investigate the transcriptome and molecular functioning of Ophiostoma floccosum and compare this to transcriptomic data available for Ophiostoma piliferum and other Ophiostoma species, O. novo-ulmi, O. clavigerum and O. piceae. This research contributes to the development of O. floccosum and O. piliferum as hosts for protein expression and advances the knowledge of gene expression and molecular functioning in this genus. To gain insight into the molecular functioning of O. floccosum, an expressed sequence tag (EST) collection from yeast-like growth (blastospores) was created during early phase growth. A total of 1207 EST sequences with an average length of 713 bp were identified. Clustering and assembly of the high-quality EST data set resulted in the identification of 598 unique putative transcripts (UPTs). Functional classification of these UPTs, using both homology searching and ab-initio methods, indicated that the majority of protein transcripts produced were involved in metabolism and cell proliferation. Up-regulation of mitochondrial transcripts involved in respiration and the presence of transcripts homologous to enzymes involved in the tri-carboxylic acid cycle indicated that aerobic respiration was likely the preferred method of ATP production in O. floccosum blastospores. However, the putative identification of genes encoding alcohol dehydrogenases within O. floccosum ESTs and the presence of homologues in other Ophiostoma species would suggest that these Ophiostoma species are also likely to be capable of metabolic functioning under anaerobic conditions. To identify homologous genes between Ophiostoma species, the O. floccosum EST data set was compared to 20,783 ESTs from other Ophiostoma species including O. piliferum, O. novo-ulmi, O. clavigerum and O. piceae. All UPTs identified within each of the datasets were aligned resulting in the identification of 347 clusters containing EST sequences from more than one Ophiostoma species. Six were identified that had homologues in all of the datasets excluding O. piceae. Three of the six homologous UPTs were predicted to function in core metabolism with two of the UPTs identified as encoding enzymes used in the glycolysis pathway and one encoding a 60S ribosomal protein. The other three homologous UPTs were thought to have a functional role in protein fate and were putatively identified as being a superoxide dismutase, heat-shock protein and a structural alpha-B chain tubulin gene. Of the 347 clusters, 86 of these contained transcripts identified in the O. floccosum EST datasets, and of these 86, only 10 fragments did not align with any significant homology to other fungal sequences contained in the NCBI non redundant database, indicating that the majority these transcripts are conserved in other fungal species. Predicted genes within the Ophiostoma EST datasets were also investigated to determine codon usage and to identify the presence of genes predicted to encode proteases. Both are important factors in recombinant protein expression. Protease production can severely inhibit the production of recombinant protein in fungal hosts. Based on sequence homology to known proteases, putative proteases were identified in all of the Ophiostoma species investigated with the exception of O. piceae. Homologues for all six peptidase groups were identified including a possible glutamic acid protease and proportionally high numbers of serine and metallo-protease homologues. This research constitutes the first reported findings of putative peptidases in the aspartic, cysteine, glutamic and threonine peptidase families in Ophiostoma species. Key to the over-expression of recombinant proteins is the optimisation of codons in a cloned gene to better utilise available tRNA species within the recombinant host. No codon bias was apparent between up-regulated and lower frequency transcripts in O. floccosum, O. piliferum, O. clavigerum and O. novo-ulmi. Codon usage was found to be consistent between these Ophiostoma species. However, a large difference between the codon usage in mitochondrially encoded genes compared to nuclear encoded genes in O. floccosum was indicated. To optimise the efficiency of a recombinant expression system, we sought to identify promoters in both O. floccosum and O. piliferum that may be applied to a vector system. Using EST data, the most up-regulated UPTs identified from O. floccosum and O. piliferum ESTs were a putative subunit 4 of the NADH-ubiquinone oxidoreductase protein (NADH-UR4) and a possible heat-shock protein (HSP), respectively. A unique hydrolase gene was also identified by molecular probing of O. floccosum genomic DNA. This putative 96 kd protein, called PLIP-Lg, was predicted to be a mitochondrial A1 phospholipase based on both nucleotide and predicted amino acid sequence structure and homology. These gene sequences were investigated using genome walking methods to further elucidate nucleotide sequences in the 5' and 3' directions. In silico investigation of the 5' promoter region of the genes identified a number of predicted transcription factor binding sites, including possible TATA boxes identified previously in the promoter region of an O. floccosum protein. Additionally, RT-PCR methods were used to compare the expression of these transcripts throughout growth in both the mycelial and blastospore forms. All three predicted genes were found to be transcribed throughout growth in both morphological forms and, thus, the use of their promoters in a vector system would not be limited to one morphology. However, the level of expression in blastospores compared to mycelial growth varied by up to 20 fold. Therefore, the morphological form of the fungi did influence the level of expression of these genes and is a factor for consideration for future promoter use. This PhD thesis research provides the first comprehensive investigation into gene expression and the transcriptome of O. floccosum while also providing the first comparative look into similarities between the transcriptomes of several Ophiostoma species. Subsequently, this research adds to the knowledge of metabolic functioning in Ophiostoma species and illustrates the usefulness of EST analysis in determining core molecular functioning within this group. Further to addressing these goals, the research will augment future research into various biotechnological applications for the genus, specifically the development of O. floccosum and O. piliferum as hosts for recombinant protein expression.

Fungal mitochondria are variable in size due to the presence of potential mobile elements such as group I and group II introns and homing endonuclease genes (HEGs). In this work the mitochondrial large ribosomal subunit gene (mt-rnl) of Ophiostoma ulmi and related species have been screened for the presence of introns and intron encoded proteins. Five introns have been noted in different regions of the rnl gene of O. ulmi and related species. Based on this rnl survey and rnl data from Genbank, an rnl intron landscape for ascomycetous and basidiomyctous fungi was generated by using bioinformatic based analysis. A total number of 23 possible intron insertion sites were found in the rnl gene of ascomycetous and basidiomycetous fungi. The results also indicate that regions of the rnl gene are more prone to intron invasion then others. The second project dealt with the evolution of mitochondrial ribosomal protein S5 (rps3) gene within the filamentous ascomycetes fungi. Within members of this group of fungi the rps3 gene typically is a component of the mL2449 group I intron but there are free-standing forms of rps3. The study examined if these free standing forms evolved only once due to an as of yet unknown recombination event or if the rps3 gene was transferred from the mL2449 intron to a new mtDNA locus several times during the evolution of the filamentous ascomycetes fungi. The third project was to sequence and characterize the intron and HEG found in the mitochondrial NADH dehydrogenase 4 (nad4) gene of an undescribed species of Pesotum. A 1.4 kb group IC2 intron has been identified in the nad4 gene of Pesotum strain WIN (M)1630. Overall the three studies demonstrate the invasive nature of introns and their associated ORFs and the potential of these introns to influence gene structure and size variation among the fungal mtDNAs.

Thèse (M.Sc.)--Université Laval, 2005. / Bibliogr.: f. 110-112. Publié aussi en version électronique.

Dissertation (PhD)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

The fungal pathogen Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) and has been responsible for the catastrophic decline of elms in North America and Europe. Double-stranded RNA (dsRNA) viruses are common to all fungal classes and although these viruses do not always cause disease symptoms, the presence of certain dsRNA viruses have been associated with reduced virulence (hypovirulence) in O. novo-ulmi. A new mitovirus was found in a Canadian isolate of O. novo-ulmi (93-1224) and has been named Ophiostoma mitovirus 1c (OMV1c). The positive strand of the dsRNA of OMV1c was 3,003 nucleotides in length and when the mitochondrial codon usage pattern was employed (mitochondria use UGA to encode tryptophan rather than as a chain terminator), a single large open reading frame (ORF) was found. This ORF had the potential to encode a protein of 784 amino acids, and revealed a high degree of nucleotide identity to genes encoding RNA-dependent RNA polymerase (RdRp) in other mitoviruses. The putative RdRp region of the newly characterized virus had the highest sequence similarity to Ophiostoma mitovirus 1b. The 5’- terminal sequence of the positive strand could potentially be folded into a double-stranded stem-loop structure with a free energy of 16.6 kcal/mol. Attempts to cure the O. novo-ulmi isolate 93-1224 of virus were unsuccessful. Screening of the re-cultured isolates for the presence of OMV1c revealed that it was still present in the fungus despite repeated hyphal tip transfer, a method known to cure cytoplasmic but not mitochondrial viruses. Based on the genome size, phylogenetic analysis, and the observation that infected isolates could not be cured, it was surmised that the virus was a member of the genus Mitovirus (family Narnaviridae). To assess the distribution of the virus in O. novo-ulmi at the disease front in Winnipeg, a small sample of thirteen isolates were screened for the presence of the new mitovirus. All proved to be negative for OMV1c, which indicated this dsRNA virus was rare and that isolate 93-1224 was the only isolate identified to date infected with OMV1c. It was also discovered that the isolate O. novo-ulmi 93-1224 potentially harboured more than one virus. Electron microscopy of fractionated cells revealed the presence of two flexuous rod-shaped particles that may represent additional novel viruses. / Graduate

Although the genetic aspects of mating are a rapidly expanding field of study, little information is available for the genus Ophiostoma. The first MAT information for the genus focussed on only three species and this was as such, hardly representative of the genus. In this study, existing DNA sequence data were used as a starting point to expand the available knowledge on mating genes to other species of Ophiostoma. Ophiostoma quercus, one of the better-studied species of Ophiostoma was the focus of the initial investigation. The heterothallic mating strategy of O. quercus was confirmed and isolates of both mating-types were used for the molecular analysis of the MAT genes. Regions of both MAT idiomorphs were observed in both mating-type isolates. This discovery was unexpected and suggests an unconventional mating organisation for O. quercus as compared to other heterothallic fungal species. Such a system is not unprecedented for fungi, but is unique for the genus Ophiostoma. The primers developed for O. quercus were tested in isolates representing 17 species of Ophiostoma. These primers were used successfully to amplify a large segment of the MAT-2 idiomorph in all isolates tested. This significantly expanded on the amount of data available for the MAT genes of Ophiostoma. Analysis showed that these isolates share a high amount of conservation in the MAT-2 open reading frame. This region of the genome is, therefore, not useful for phylogenetic analyses. However, the availability of primers for the region might facilitate testing of other areas of the full idiomorph for phylogenetic inference. Overall, the results presented in this study represent a significant increase in the knowledge available on MAT genes in Ophiostoma. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Genetics / Unrestricted

Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Aug. 12, 2008. Includes bibliographical references.

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