Location: Sunflower Research
Project Number: 5442-21220-028-27
Start Date: Jun 01, 2014
End Date: Sep 30, 2015
The experiments will utilize massively parallel DNA sequencing technology performed on an Illumina sequencing platform to examine the “interactome” between S. sclerotiorum and pea over a time course during compatible (susceptible) and incompatible (partially resistant) interactions. The sequencing of cDNA tags has been demonstrated to be subject to less background noise than traditional hybridization based (i.e. microarray) approaches. We will use a previously developed EST data set to aid interpretation of sequence reads from the Illumina platform as well as public EST database resources of pea, closely related model species and the S. sclerotiorum genome sequence. The expression profiles and the difference between susceptible and partially resistant pea genotypes and the difference that occurs over time will be used to examine the expression of genes from host and pathogen during their interaction. We will also use staining techniques and microscopy to examine the host response to infection. Pea stem sections will be stained with phloroglucinol-HCl and Maule reagents to indicate lignin and S monomer of lignin contents, respectively, and visualized by light microscope. Additionally, the total lignin and lignin monomer contents will also be determined using the analytical cell wall compositional platform available at the Great Lakes Bioenergy Research Center at MSU. This approach will allow us to dentify and quantify pathogenicity genes expressed by S. sclerotiorum as well as genes participating in the resistance pathway in pea. ESTs of interest will also be examined by analysis of gene ontology classes. Tissue will be collected from infected plants over a time-course after inoculation to capture the initial, sustained, and late gene expression changes associated with the interaction. It is expected that we will identify genes and pathways involved in the pathogenicity of S. sclerotiorum on pea and genes and signaling pathways involved in the partial resistance of pea to Sclerotinia. Additionally, by identifying genes involved in resistance we plan to develop genetic markers for marker assisted breeding of resistant pea lines. The sequencing expression profiling work will be confirmed with real-time PCR assays for a select number of transcripts. We have already sequenced one expression profiling experiment and will use the results of this current analysis to guide future direction, such as additional time points or cultivars.