Location: Sunflower Research
Project Number: 5442-21220-028-01
Start Date: Jul 01, 2010
End Date: Jun 30, 2014
The proposed study will use a next-generation high throughput sequencing approach for identification host and pathogen of genes differentially expressed during infection of resistant and susceptible canola (Brassica napus) lines with Sclerotinia sclerotiorum. This will be followed by comparative analysis for functional assignment and confirmation of expression patterns through quantitative real-time procedures. B. napus is very closely related to the Arabidopsis thaliana which is the most well characterized plant species with a whole genome sequence available. Therefore, use of B. napus in this study will greatly increase the chances of identification of white mold resistance genes and pathways. Previous research supported by the Sclerotinia Initiative conducted at NDSU has led to the development of a double haploid homozygous progeny from a PI line of B. napus with significant resistance to white mold. Progeny from the same line with almost no resistance to the disease are also available. These two lines will serve as material to initiate screening for resistance genes. The approach will be to inoculate the resistant and susceptible varieties of canola with aggressive isolates of S. sclerotiorum, collect RNA at different time points after inoculation, pool the total RNA from these different time points and sequence the pooled RNA using Illumina sequencing. This next-generation Illumina/Solexa based sequencing method generates millions of short reads (75bp) which can then be anchored to the A. thaliana and S. sclerotiorum genome sequences respectively. EST information from these specific reactions will be used to conduct in-silico comparisons to identify plant genes involved in resistance to S. sclerotiorum. Fungal genes identified will also be used for analysis similar to that stated for plants, to identify fungal genes (including pathogenicity genes) expressed/ repressed during all host-pathogen interactions. Selected ESTs from this study representing resistance or pathogenicity genes would be used for qRT-PCR to confirm enhanced expression during disease development and to identify the infection stage when they are expressed.