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United States Department of Agriculture

Agricultural Research Service

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Research Project: Identification of Resistance and Pathogenicity Genes Associated with Sclerotinia Sclerotiorum Infection Using Next-Generation Sequencing

Location: Sunflower Research

2013 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.

A comprehensive understanding of the molecular events occurring during infection of canola by the phyto-pathogenic fungus, S. sclerotiorum will likely generate novel information regarding the white mold disease and may lead to more effective disease control measures. The primary objective of this study is to identify differences in expression patterns of host and pathogen genes during infection of canola lines either susceptible or tolerant to S. sclerotiorum using next-generation Illumina/Solexa sequencing. Insilica analysis of the resulting data for determination of gene identities will be followed by validation of expression pattern differences using quantitative real-time PCR techniques. This will enable identification of potential resistance and pathogenicity genes in the host and the fungus respectively.

The S. sclerotiorum isolate used for genome sequencing, NE152 (also known as 1980) was obtained from collaborators at the University of Nebraska for use in our studies. Experiments were conducted with the double haploid canola lines differing in their susceptibility to white mold. During the initial period of limited seed stocks, preliminary experiments were conducted to test the efficacy of the proposed approach in identifying genes. During this preliminary trial in which a small number of plants belonging to the resistant (NEP 63) and susceptible (NEP32) lines were inoculated with a highly aggressive S. sclerotiorum isolate (NE152) in a growth chamber, an established petiole inoculation technique involving potato dextrose agar (PDA) plugs with actively growing S. sclerotiorum was utilized and samples were collected at 24 and 48 hours post inoculation. Non-inoculated, control canola plants were treated with PDA plugs containing no fungus. At both the time points examined, there were no observable phenotypic differences between the susceptible and resistant canola lines and there was no difference in appearance of control petioles. However, four days post inoculation the plants from the susceptible line died whereas the resistant plants continued growing unharmed. Inoculated petioles were harvested at 24 and 48 hours post inoculation and immediately flash frozen in liquid nitrogen to prevent degradation of genetic material and were stored at -80°C. RNA extraction, mRNA purification, and cDNA library preparation was conducted for these samples using commercially available kits. The prepared libraries were submitted to the DNA Sequencing and Analysis Facility, Bio Medical 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, a 76 cycle run was completed. This initial sequencing run was performed using the pooled samples in a single lane to validate the library preparation process. This initial run generated more than two million sequence reads. After validating the library preparation protocol, large scale experiments involving more time points were conducted using two different inoculation methods, the petiole inoculation method conducted previously and a leaf based inoculation method.

This project addresses stated Sclerotinia Initiative goals including, "Pathogen and Host Genomics" as well as "Pathogen Biology and Development". One of the anticipated outcomes of this project is the identification of genes mediating pathogenicity in S. sclerotiorum and resistance in canola. The identification of these genes is likely to have relevance to other important crops that are susceptible to S. sclerotiorum infection (e.g., dry beans, soybeans, pea, and sunflower.) The data produced in this project could also lead to the generation of new functional molecular markers associated with white mold resistance which would be valuable in future molecular marker assisted breeding programs to produce agriculturally-significant crops with increased resistance to S. sclerotiorum.

Last Modified: 7/28/2014
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