Towards Control of Dutch Elm Disease: dsRNAs and the Regulation of Gene Expression in Ophiostoma novo-ulmi




Carneiro, Joyce Silva

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Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) which has had a severe impact on the urban landscape in Canada. This research program focused on developing molecular genetic strategies to control this pathogenic fungus. The first strategy involved the development of RNA interference (RNAi) for the down-regulation of genes involved in pathogenicity. An efficient RNAi cassette was developed to suppress the expression of the endopolygalacturonase (epg1) locus which encodes a cell-wall degrading enzyme. This epg1-RNAi cassette significantly reduced the amount of polygalacturonase activity in the fungus and resulted in almost complete degradation of epg1 mRNA. The need for a native promoter to selectively down-regulate specific gene loci was addressed by developing a carbon-catabolite regulated promoter (alcA) to drive the expression of the epg1-RNAi cassette. The expression of an alcA-driven epg1-RNAi cassette resulted in the down-regulation of epg expression under glucose starvation but normal levels of expression in high glucose. The expression could therefore be controlled by culture conditions. The second strategy explored the potential of using dsRNA viruses to vector disruptive RNAi cassettes. An isolate of O. novo-ulmi strain 93-1224 collected in the city of Winnipeg, was infected by two dsRNA mitoviruses which upon sequence characterization were named OnuMV1c and OnuMV7. To assess the transmissibility of this dsRNA virus the infected isolate 93-1224 was paired with three naive isolates of the related fungi O. ulmi and O. himal-ulmi. Through the use of nuclear and mitochondrial markers it was determined that the virus OnuMV1c may not rely on mitochondrial fusion for transmission but may have a cytoplasmic transmission route. This investigation of gene expression and manipulation has provided tools to help understand gene regulation in O. novo-ulmi. It has also added to our knowledge of mitoviruses, their transmission and potential use as a biological control. By enhancing our understanding of transmissible hypovirulence this work contributes to efforts to develop a new approach to target DED as well as a potential model for the control of other fungal diseases.



Ophiostoma novo-ulm, Dutch elm disease (DED), RNA interference, Polygalacturonase, Alcohol dehydrogenase promoter, Gene expression, Phytopathogen, Cell wall degrading enzymes, Pectin enzyme, Mitoviruses, Pathogenicity, Virulence, Double-stranded RNA (dsRNA), hyphal anastomosis, group I and II introns, Homing Endonuclease Genes (HEGs), Single Sequence Repeats (SSRs)