Location: Sunflower and Plant Biology Research2011 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. Our cooperators at the Michigan State Research Technology Support Facility through collaborations also have access to a large number of pea ESTs that are not publicly available. 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 Sclerotinia sclerotiorum in pea and genes and signaling pathways involved in the partial resistance in pea. ADODR monitoring activities to evaluate research progress included phone calls, meetings with the cooperator, and an annual meeting held each year in January. A 35Mb expressed sequence tag (EST) data set was developed from S.sclerotiorum infected pea tissue with massively parallel sequencing on a 454 Roche platform; 145,049 reads, each averaging >200 nucleotides. Data were trimmed, based on sequence quality and adaptor sequence removal, and 128,720 reads were assembled into 10,158 contiguous sequences (contigs); 51 contigs were discarded due to high similarity to viruses and viroids. To separate S. sclerotiorumcontigs from Pisumsativum reads we utilized a tBlastx search algorithm against a fungal genome library (Sclerotinia sclerotiorum, Botrytis cinerea, Chaetomiumglobosum, Fusariumgraminearum, Magnaporthegrisea, Neurosporacrassa, and Verticillium dahlia) and a legume genome library (Glycine max, Lotus japonicus and Medicagotruncatula). This method was first developed and tested with a set of pea and Sclerotinia ESTs downloaded from public databases (NCBI and BROAD). On the test data set the tBlastx parsing method demonstrated that 90% of ESTs could be assigned with a very low (0.1%) false allocation rate. The tBlastxparsing method was applied to our 10,107 EST contigs; 53.8% of contigs were assigned to pea, 27.4% to S. sclerotiorum, 5.4% of reads were ambiguous with hits to both genome libraries, and 13.4% were unassigned. The EST contig assignment method was validated by selecting 50 pea and 50 S. sclerotiorumassigned ESTs at random, PCR primers were designed to these ESTs and PCR was run against cDNA from pea infected with S. sclerotiorum, pea only and S. sclerotiorum only. All 50 pea EST primers amplified the same size amplicons from pea infected with S. sclerotiorum and pea cDNA, indicating correct assignment of reads. Likewise, the S. sclerotiorum EST primer sets amplified 47 out of 50 predicted amplicons. Two S. sclerotiorum ESTs only amplified correct sized PCR products from the pea-Sclerotinia cDNA sample, perhaps indicating that this transcript is only expressed during the interaction with pea. One Sclerotinia primer set failed to amplify from any template.We are currently completing analysis of the 454 EST data set for publication, including gene ontology analysis to further characterize the EST data set. Simple sequence repeat (SSR) markers identified in the pea ESTs are also being screened for polymorphism among pea parental lines as additional markers for mapping studies. Once this study is published the EST data sets will be made publicly available for the Sclerotinia and pea community.