Location: Sunflower and Plant Biology Research2013 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. We have been utilizing next generation sequencing technologies to investigate the host-pathogen interaction between P. sativum and S. sclerotiorum. In initial experiments a large expressed sequence tag (EST) data set was developed with massively parallel sequencing on a 454 Roche platform. Post-trimming, the data set consisted of 145,049 reads with an average read length of >200 nucleotides. This data and EST resource was analyzed and a manuscript published in BMC Genomics 13:668. The manuscript also described a method for distinguishing pea and S. sclerotiorum sequences without a reference pea genome. Additionally SSR (microsatellite) markers were identified in the pea EST data pea and primers were developed to screen these markers for polymorphism across pea parents from 4 recombinant inbred lines, developed by our NDSU collaborator, and additional pea germplasm. A manuscript describing these novel SSR-EST markers was published in Applications in Plant Sciences 1( 1 ):1200249. The parents of a pea mapping population that our NDSU collaborator established are being utilized in a RNA-seq approach to examine the expression profile of the Sclerotinia-pea host pathogen interaction. The parents, Lifter a susceptible cultivar and PI240515 a partially resistant line were inoculated with S. sclerotiorum and the genes expressed by both the pathogen and the host were determined. Around 300 million pairs of reads were produced with a 75 bp paired-end sequencing method. As a reference genome is not available for pea we are utilizing software called Trans-ABySSand Trinity which allows de novo assembly and analysis of RNA-seq data without a reference genome. The expression profile of both pea and S. sclerotiorum are being examined at multiple time points to identify genes involved in the resistance response as well as S. sclerotiorum genes involved in pathogenicity. The RNA-seq approach has generated a large amount of transcript sequence data for each pea parent, which will be valuable for the development of additional gene-linked markers such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) which we plan to utilize in LuminexxMAP technology (or similar technologies) to map resistance to Sclerotinia in pea. We are currently preparing manuscripts describing the transcriptome characterization and gene expression profiling in pea with partial resistance to S. sclerotiorum and the expression profiling of the pathogen during infection of susceptible and partially resistant pea lines. We are also carefully examining the infection process and plant response with histological studies. Our studies have contributed significantly to the development of “crop germplasm resources and genetics” and “pathogen and host genomics”. Data generated from the initial EST analysis is available for use by the Sclerotinia and pea community and data from the current RNAseqefforts will be available shortly. The project has already developed 37 SSR markers which have been incorporated into mapping populations by our NDSU collaborator and an additional 541 SSRs have been made available.