Location: Sunflower and Plant Biology Research2012 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. 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. Plants were maintained in a greenhouse and bagged during flowering to prevent cross pollination, and seeds were harvested once seed pods were sufficiently mature. Due to 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. From the sequencing runs, a total of 153,851,612 and 109,899,384 quality filter passed 76 bp short reads were generated from petiole inoculated and leaf inoculated libraries respectively. From the inoculated libraries, reads from fungal origin (pathogen) were filtered out by aligning to S. sclerotiorum genome using software program Bowtie. The remaining reads were considered to be plant origin. Initially it was proposed to use A.thalianagenome as a reference for aligning short reads generated from sequencing, but from the preliminary analysis it was found that only very low number of reads from the libraries had matches with A. thaliana which necessitated searching for alternate sources to use as a reference. Ultimately, the Brassica EST assembly, 95k unigene set from the Brassica genome gateway (http://brassica.bbsrc.ac.uk/ ), which represents all the publicly available ESTs from A, B, and C genomes of Brassica spp., was used as reference to anchor reads. Read counts that were uniquely aligned (only one best match in the reference) were obtained; reads with more than one best match in the reference were excluded. Read count data obtained from the alignment of libraries was used to determine differential expression patterns using software package DESeq. Analysis of the massive data set generated from sequencing is in progress. So far, comparisons have been made to determine genes or ESTs differentially expressed in resistant line NEP63 compared to the susceptible line NEP32 during infection process. Preliminary analysis of the petiole inoculation libraries revealed a total of 4350 differentially expressed ESTs, of which 1946 are up-regulated. Interestingly, more than 400 ESTs seem to be turned on exclusively in resistant line, 436 ESTs are up-regulated by 10 fold change. Functional categorization of these differentially expressed genes is essential to understand the mechanism underlying the differences in resistance response in the two canola lines. Efforts in this direction are in progress. Initial searches suggest that many of the ESTs have no known function, and thus can be considered as potential candidates for identification of novel resistance genes. From the preliminary analysis of the leaf inoculated libraries, a total of 477 ESTs were found to be differentially expressed in the resistant line NEP63, 320 of them were up-regulated and 157 were down-regulated. However, from the leaf inoculated conditions no genes that are exclusively turned on in resistant line have been found in the initial analysis. Functional categorization of the up-regulated gene set was performed using Blast2Go. Of the 320 ESTs only 163 have a known role in biological processes. 24 of the 163 ESTs were found to be involved in defense response. Response to stress, hormone mediated signaling pathways, transcription factors were other categories of ESTs that may be involved in regulation of a resistant response. Analysis of petiole inoculation libraries has also been initiated. Comparison of gene expression between inoculated and non-inoculated conditions will provide an insight into the various responses of the host to pathogen infection. Additionally, comparison of fungal genes that are expressed during infection process and in culture will allow us to gain understanding of mechanisms employed by S. sclerotiorum during pathogenesis. Analysis of the above two mentioned comparisons have been initiated and continue at present. Our results will contribute to the development of new functional molecular markers associated with white mold resistance that will have utility in plant breeding programs.