Location: Sunflower and Plant Biology Research2011 Annual Report
1a. Objectives (from AD-416)
Elucidation of differential host and pathogen gene expression during resistance and susceptible interaction between Sclerotinia sclerotiorum and its host using Brassica napus as a model system.
1b. Approach (from AD-416)
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.
3. Progress Report
This project was initiated on July 1, 2010, research is ongoing, and the overall objective is the identification of resistance and pathogenicity genes associated with Sclerotinia sclerotiorum infection using next-generation sequencing. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. The primary objective of the project is to characterize the differential gene expression patterns between canola (Brassica napus) lines susceptible and resistant to infection by Sclerotinia sclerotiorum using next-generation, Illumina-based DNA sequencing to identify putative resistance genes. Selected doubled haploid canola lines (NEP 32 and NEP 63) differing in their susceptibility to white mold disease used in this study were generated at NDSU from B. napus PI Ames 26628 in a previous project supported by the Sclerotinia Initiative, and the initial months of the current project were devoted in part to increasing bulk seed suppliesfor subsequent planned experiments. Plants were maintained in a greenhouse and bagged during flowering to prevent cross pollination, and seeds were harvested once seed pods were sufficiently mature. During this initial period of limited seed stocks, preliminary experiments were conducted in which a small number of plants were inoculated with a highly aggressive S. sclerotiorum isolate (NE162) in a growth chamber for each of the selected time points (i.e., 8, 16, 24, and 48 hours post inoculation); susceptible line (NEP 32) compared to resistant line (NEP 63). Harvested petioles were flash frozen in liquid nitrogen to prevent degradation of genetic material and were stored at -80°C. For the samples from the 24 and 48 hour post inoculation time points, RNA extraction, mRNA purification, and cDNA library preparation were completed using commercially available kits. The prepared libraries were submitted to the DNA Sequencing and Analysis Facility, BioMedical Genomics Center at the University of Minnesota for next generation sequencing using the Illumina GA IIx instrument. After passing an initial quality control KAPA qPCR assay, libraries were pooled and a single read, 76 cycle run was completed. This initial run generated more than two million sequence reads, which is perhaps a little lower than expected. The percentage of these sequence reads that correspond to plant genes is currently being determined, although it should be pointed out that at the time points analyzed (i.e., 24 and 48 hours post inoculation) the majority of the inoculated petioles were badly damaged by S. sclerotiorum and it is possible that much of the plant RNA has degraded. Our results will contribute to the development of new functional molecular markers associated with white mold resistance with utility in plant breeding programs.