Location: Sunflower and Plant Biology Research2010 Annual Report
1a. Objectives (from AD-416)
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 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. 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 individuals from 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.
1b. Approach (from AD-416)
The experiments will utilize massively parallel sequencing technology performed on an Illumina GA2 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 an EST data set that we are currently analyzing to aid interpretation of sequence reads from the Illumina GA2 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. 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.
3. Progress Report
This project was initiated on June 1, 2009, research is ongoing, and the overall objective is to identify genes and biochemical pathways involved in the pathogenicity of S. sclerotiorum on pea and genes and signaling pathways involved in the partial resistance of pea to Sclerotinia. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. Support for genomics assisted breeding for Sclerotinia white mold. The fungus Sclerotinia sclerotiorum, is a devastating disease of many crop plants and has the potential to cause significant economic losses in pea. The objective of this research is to examine the mRNA expression profile of the pea-Sclerotinia interaction between two pea genotypes (susceptible and partially resistant) over three time points. To do this, we are utilizing the Illumina GA2 platform, a new massively parallel sequencing platform capable of sequencing a total of 160-170 reads per run. The sequencing reactions are divided into 8 lanes, each lane is capable of reading in excess of 24 million reads. To the best of our knowledge, is the first attempt to utilize the Illumina GA2 massively parallel sequencing platform for mRNA expression profiling in Sclerotinia and one of the few studies to simultaneously examine host and plant pathogen mRNA profiles using massively parallel sequencing. We currently have a large (~35Mb) EST data set generated on the P. sativum- S. sclerotiorum interaction which was funded previously by the National Sclerotinia Initiative. We are utilizing our EST data set and other publicly available EST data sets to interpret the mRNA expression profiles that we generate on the Illumina GA2. Research on gene expression can provide insights into the molecular mechanisms controlling pathogenicity and host resistance. 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. By identifying genes involved in resistance we will be able to develop markers for marker assisted breeding of resistant pea lines, and identify target genes for future functional research using reverse/forward genetics and proteomics, that will eventually facilitate development of novel management strategies for control of white mold disease of pea.