Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-028-27-S
Project Type: Specific Cooperative Agreement
Start Date: Jun 1, 2014
End Date: Sep 30, 2017
Sclerotinia white mold, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a devastating disease of many crop plants and can cause significant economic losses in dry pea under the appropriate environmental conditions. The interaction between pathogen and host and the expression of host resistance may depend strongly on specific interactions between S. sclerotiorum and the pea host (Pisum sativum). It is important to develop genomic resources for S. sclerotiorum and pea that are relevant to the interaction between S. sclerotiorum and P. sativum. Currently, little is known about the genetic mechanisms that control the basic biology and pathology of S. sclerotiorum interacting with pea. Although we have a S. sclerotiorum genome sequence, few pea genomic resources exist. By conducting these sequencing studies we will generate pea genomic resources that will be a valuable resource for current and future studies. The objective of this proposal is to utilize recent advances in massively parallel sequencing technology to study the regulation of genes during the S. sclerotiorum and P. sativum host-pathogen interaction. To identify pea resistance genes and Sclerotinia pathogenicity genes we will compare gene expression in parents of a pea recombinant inbred line that is segregating for partial resistance to S. sclerotiorum. The identification of genes involved in partial resistance may lead to the development of markers for marker assisted breeding for resistance to S. sclerotiorum. The expression profiling studies will also provide valuable information on the genetics of the interaction between host and pathogen which may lead to further insights and solutions for developing resistant material not only in pea but in other crops affected by Sclerotinia spp. Additional objectives of this proposal are to provide characterization of genes for improved annotation of the Sclerotinia genome project and gene function discovery for both pea and S. sclerotiorum.
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 identify 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.