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

Dutch elm disease (DED) is a debilitating wilt disease that has decimated elm populations globally. The current pandemic of this disease is caused by the ascomycete fungal pathogen Ophiostoma novo-ulmi. A number of strategies have been used to attempt to mitigate the effects of DED but none have met any sustainable success, and the disease continues to have severe ecological and economic impacts. Consequently, research focus has turned to the development of control strategies at the genetic level. One such genetic strategy is the use of naturally occurring fungal viruses (mycoviruses) to induce hypovirulence in their fungal hosts. Hypovirulence, or attenuation of fungal pathogenicity using mycoviruses, has been well studied in other systems but has yet to be developed for O. novo-ulmi. A candidate virus, OnuMV1c, was found in an isolate of O. novo-ulmi (93-1224) at the western Canadian disease front and its genome was sequenced. OnuMV1c is a mitochondrial virus and has a 3.1 kb single-stranded positive RNA genome that encodes an RNA-dependent RNA polymerase (RdRp) involved in its replication as a double-stranded RNA molecule. It exists in O. novo-ulmi mitochondria in both its single-stranded and double-stranded forms. Our research group identified OnuMV1c as a potential candidate for biological control of Dutch elm disease. Our long-term research goal is to use the virus as a means to activate the RNA interference pathway of O. novo-ulmi, leading to down-regulation of genes involved in pathogenicity. If OnuMV1c is engineered such that it carries an RNA interference cassette in addition to its own complement of genes, it could act as an enhanced hypovirus. RNA interference (RNAi) is a cytoplasmic process, and therefore in order to use OnuMV1c for RNAi the viral genome needed to be reprogrammed such that it could be expressed in the cytoplasm rather than the mitochondria. The objectives of my master’s research were to 1) genetically engineer OnuMV1c to express in the cytoplasm using a cDNA reverse genetics approach, and 2) test the functionality of the re-engineered cDNA OnuMV1c virus (MV1cCyt). The first objective was accomplished by modifying codons in the RdRp sequence of OnuMV1c such that the sequence could be translated in the cytoplasm. This genetically engineered cytoplasmic version of OnuMV1c, named MV1cCyt, was flanked with exogenous promoter and terminator sequences to drive its transcription. The entire construct was engineered as a cDNA molecule and was cloned into the fungal transformation vector pAN7-1, which was used to transform O. novo-ulmi protoplasts. The second objective was achieved through the use of strand-specific RT-PCR, a technique that allowed the detection of both the positive and negative strands of MV1cCyt. Results indicated that while four individual cell lineages contained MV1cCyt cDNA stably integrated into the nuclear genome, only one transformant was able to produce double-stranded MV1cCyt RNA. These results have important implications for the use of OnuMV1c as an engineered hypovirus and represent the first step towards the development of a biological control strategy for Dutch elm disease. / Graduate