2012 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.
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.
Achievements to date both directly advance our ability to improve pea genotypes for resistance to white mold (i.e. development of additional markers for marker assisted breeding) and set the ground work for the identification of novel resistance genes and improved understanding of this host-pathogen interaction.
First, a 454 sequence data set derived from the pea-S. sclerotiorum interaction was analyzed and annotated. We found 597 pea ESTs which are putatively involved in plant defense and response to biotic or abiotic stress,and 155 S. sclerotiorum ESTs involved in pathogenicity or virulence. To assist in the development and refinement of pea linkage maps, 37 EST-derived SSR markersfrom the 454 sequence data were developed and screened for 23 pea individual cultivars including parents of four Pisumsativum recombinant inbred line (RIL) mapping populations developed for improved white mold resistance. Eleven SSR markers produced polymorphism in at least one population.
To further investigate the interaction and quantify gene expression over time global gene expression profiling of partially resistant and susceptible pea lines infected with S. sclerotiorum was conducted using an Illumina GA2 sequencing platform with a 75 bp paired end sequencing protocol. Pea stem samples from a susceptible pea cultivar ‘Lifter’ and a partially resistant cultivar ‘PI240515’either inoculated with S. sclerotiorum or mock–inoculated were collected at 12, 24 and 36 hour after inoculation. Additionally, RNA was extracted from S. sclerotiorum growing on the culture medium used for inoculation. After RNA extraction, cDNA library preparation and barcoding, a total of 30 cDNA samples were sequenced in 7 lanes of one Illumina Genome Analyzer flowcell, which produced more than 300 million paired-end reads. After first-round de novo assembly, those reads resulted in 60,656 Lifter contigs, 67,893 PI240515 contigs and 18,178 S. sclerotiorum contigs obtained from mock-inoculated samples with contigs larger than 200 nucleotides; 71,228 contigs and 81,533 contigs were assembled from the Lifter and PI240515 samples infected with S. sclerotiorum. A second round of de novo assemble was executed for the gene expression profiling study between two cultivars. All Lifter-Sclerotinia related reads were assembled into 61,056 contigs larger than 300 nucleotides and all PI240515-Sclerotinia reads into 65,047 contigs. Based on the tBLASTx method we developed previously and using the first-round contigs from mock control as reference, the second-round Lifter-Sclerotinia contigs were sorted into 44,998 pea ESTs, 14,436 S. sclerotiorum ESTs, 360 ambiguous ESTs and 1,262 ESTs cannot be assigned. The second-round PI240515-Sclerotinia contigs were sorted into 48,174 pea, 14,513 S. sclerotiorum, 383 ambiguous and 1,977 unassigned.
By comparing the contigs from the 2 pea cultivars, 541 simple sequence repeat (SSR) markers from pea were identified. These SSR markers have been provided for the construction of pea linkage maps. All pea ESTs from Lifter and the PI line were annotated based on Arabidopsis, Medicago and Soybean annotation databases, respectively. A total of 20,526 annotated pea ESTs (average length 1,454 nucleotides) with 95% similarity in the two cultivars have been selected for comparison of gene expression profiling. 10,596 S. sclerotiorum ESTs (average length 1,767 nucleotides) were also selected and will be used to determine the expression profiling of the fungus during this host-pathogen interaction.
Through gene expression profiling study, we hope to identify genes and pathways involved in resistance to facilitate the breeding of pea lines with a higher level of resistance to white mold. We also plan to identify S. sclerotiorum genes with putative roles in infection, growth, and pathogenicity and to help us better understand the mechanism of this pathogen.
In summary, during 2011, an expressed sequence tag (EST) resource of pea and S. sclerotiorum was characterized and a publication prepared for submission. From this EST resource 37 simple sequence repeat (SSR) markers for pea breeding were developed and a manuscript describing this was submitted for publication. A time course gene expression profiling study of S. sclerotiorum on pea was conducted with RNA seq, which produced approximately 300 million paired end reads and this RNA-seq data set was mined for an additional 541 SSR markers for use in the development of linkage maps for S. sclerotiorum resistance breeding.