|Institution:||University of Alberta|
|Department:||Department of Agricultural, Food and Nutritional Science|
|Full text PDF:||http://hdl.handle.net/10048/410|
Sclerotinia sclerotiorum is a devastating pathogen causing stem rot in Brassica napus (canola). Microarray analysis was performed to investigate pathogen-induced transcript profiling in B. napus responses to S. sclerotiorum. Several genes were identified, which included defensins, those encoding proteins involved in oxidative burst, the biosynthesis of jasmonic acid (JA), and several transcription factors (TFs), but not salicylic acid (SA)-related genes. To further characterize the roles of SA, JA and ethylene (ET) in the response of B. napus and Brassica carinata (tolerant species) to S. sclerotiorum, the expression of five genes known to respond to these phytohormones were investigated. We observed that S. sclerotiorum triggered JA/ET signaling in B. napus. Furthermore, the heterologous expression of 1-aminocyclopropane- 1-carboxylate (ACC) deaminase, which reduced ET levels, enhanced the susceptibility of B. napus to S. sclerotiorum. Our microarray analysis also revealed the importance of TFs in mediating responses of B. napus to S. sclerotiorum. To probe the involvement of one such TF family in B. napus, the WRKYs, public sequence databases were mined. Three groups of B. napus WRKYs were indentified from a phylogenetic tree and four selected ones were shown to localize to the nucleus using GFP fusions. Sclerotinia sclerotiorum and Alternaria brassicae (another necrotrophic pathogen affecting canola) and phytohormone-induced expression of representative WRKYs from each clade was also investigated using quantitative real-time PCR. In another aspect of our study, two recombinant single chain variable fragment (ScFvs) antibodies specific for a S. sclerotiorum endopoly-galacturonase (SSPG1d) were isolated and characterized. Our results indicated that these antibodies may have utility in the detection of this pathogen when used with other S. sclerotiorum-specific antibodies in a panel format assay. Transgenic Arabidopsis expressing the ScFvs were evaluated for tolerance to S. sclerotiorum and it was observed that the heterologous expression of the cDNAs encoding these ScFvs did not enhance the tolerance of Arabidopsis. However, additional research aimed at stabilization of the ScFvs and/or their localization must be conducted together with research into their usefulness in imparting tolerance in B. napus to this pathogen, since the observed effects in this species may be different from A. thaliana.