Progress 08/24/04 to 06/30/09
Outputs
Progress Report Objectives (from AD-416) Establish an EST library of Sclerotinia sclerotiorum and sequence about 4000 EST clones. Establish an interaction EST library of Pisum sativum tissue infected with S. sclerotiorum and sequence about 2000 EST clones. Develop a procedure using chemical mutagenesis to generate mutant strains of S. sclerotiorum that are non-pathogenic on grain legumes. The mutant strains will be used to generate an EST library. Approach (from AD-416) The strain WM1 of S. sclerotiorum collected from pea field will be used to construct a cDNA library. Similarly pea cultivar Franklin will be inoculated with white mold and the infected host tissue will be used to isolate cDNA for library construction. Total RNA from the mycelium and from the infected host tissue will be isolated and purified using the oligotex mRNA isolation kit. A cDNA library will be prepared in plasmid pSPORT1 using the superscript plasmid system for cDNA systesis and cloning. After plasmid ligation, they will be transformed into E. coli. Individual colonies will be selected and transferred to 96-well microtiter plates and used for automatic DNA sequencing. Chemical and physical mutagenic agents, such as 5-bromouravil, N-methyl- N'-nitro-N-nitrosoquanidine, and ultraviolet light, will be used to generate mutants of S. sclerotiorum and the mutants will be screened for pathogenicity. Non-pathogenic mutants will be used in cDNA library construction. Documents SCA with WSU. Formerly 5348-21000-014-04S (12/08). Significant Activities that Support Special Target Populations Total RNA was isolated from pea tissues infected with Sclerotinia sclerotiorum in order to develop expressed sequence tags for both the pathogen and the host. After confirming RNA quality and presence of transcripts from both the plant and the pathogen, messenger RNA was isolated from the total RNA by poly-A separation. The mRNA sample was converted into a �normalized� cDNA pool, which reduced the abundance of highly expressed transcripts to increase chances of sequencing rare transcripts. The cDNA pool was run on a massively parallel sequencing, 454-GS FLS pyrosequencing platform, to obtain more than 40 Mb of sequence data, which corresponds to approximately 50,000 clones of a traditional cDNA library. Analysis of the EST data will generate a substantial number of genes involved in pathogenicity and resistance response. The results will be used to conduct expression profiling of the Sclerotinia- pea interaction and will be useful to refine the annotation of the Sclerotinia genome and for marker development in pea. The cooperator�s performance is monitored by regular visits to the cooperator�s research facilities, which are located at Washington State University. During these visits the project plan is discussed, program goals are confirmed, and data is analyzed and interpreted with respect to realizing the objectives of the research program.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Establish an EST library of Sclerotinia sclerotiorum and sequence about 4000 EST clones. Establish an interaction EST library of Pisum sativum tissue infected with S. sclerotiorum and sequence about 2000 EST clones. Develop a procedure using chemical mutagenesis to generate mutant strains of S. sclerotiorum that are non-pathogenic on grain legumes. The mutant strains will be used to generate an EST library. Approach (from AD-416) The strain WM1 of S. sclerotiorum collected from pea field will be used to construct a cDNA library. Similarly pea cultivar Franklin will be inoculated with white mold and the infected host tissue will be used to isolate cDNA for library construction. Total RNA from the mycelium and from the infected host tissue will be isolated and purified using the oligotex mRNA isolation kit. A cDNA library will be prepared in plasmid pSPORT1 using the superscript plasmid system for cDNA systesis and cloning. After plasmid ligation, they will be transformed into E. coli. Individual colonies will be selected and transferred to 96-well microtiter plates and used for automatic DNA sequencing. Chemical and physical mutagenic agents, such as 5-bromouravil, N-methyl- N'-nitro-N-nitrosoquanidine, and ultraviolet light, will be used to generate mutants of S. sclerotiorum and the mutants will be screened for pathogenicity. Non-pathogenic mutants will be used in cDNA library construction. Documents SCA with WSU. Significant Activities that Support Special Target Populations This is a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. Research efforts in this agreement were focused on two objectives: 1) the construction of cDNA libraries from the interaction of the ascomycete fungus Sclerotinia sclerotiorum with pea, and 2) determining the nature of putatively symbiotic bacteria associated with S. sclerotiorum. Three cDNA libraries were generated from culture-grown mycelium on PDB and YPG media and transformed into E. coli. Sequences from each of these EST libraries and most clones had similarity to bacterial sequences from Pseudomonas spp. despite no evidence for contamination of the growth medium. When the growth medium was changed to YPG, the ratio of sequences with similarity to bacterial genes relative to that of fungal sequences was reduced. This result suggests the presence of symbiotic bacteria in S. sclerotiorum strain WMA1. We detected the occurrence of bacteria in several S. sclerotiorum strains by PCR with bacterium-specific primers. Primers (63F and 1378R) were designed to bacterial 16S ribosomal DNA. This primer set amplified a fragment only from WMA1 and not from strain WMA1-5 and strain 1980 from pea and bean. Strain WMA1-5, obtained by sub- culturing WMA1 on gentamicin, and strain 1980 from University of Florida (in culture for many years), were bacteria-free based on PCR. Sequences of fragments amplified by the bacteria-specific primers had the greatest similarity to Ralstonia spp. These results suggest that strain WMA1 may harbor symbiotic bacteria which are absent from strain 1980 and some subcultured strains. The biological significance of these putatively symbiotic bacteria is being investigated. Research progress is monitored via informal discussions (several times a week), monthly progress and planning meetings, and regular visits to the collaborator's laboratory facilities, which are co-located with the ARS at Washington State University, Pullman, WA.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This is a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. The project is aimed at developing cDNA libraries for expressed genes (expressed sequence tags) of the pea pathogen Sclerotinia sclerotiorum and interactions between the pathogen and the host. After selecting an isolate WM-A1 representative of the pathogen population based on a number of molecular markers and phylogenetic analysis, RNAs were isolated from this isolate and cDNA libraries were constructed. Sequencing selected clones of the library has confirmed many known and some previously unreported genes from S. sclerotiorum. Cytology of this isolate was also investigated using a variety of staining techniques. The isolate was also used
to inoculate pea cultivar Lifter and procedures for optimizing isolation of RNAs from infected pea plants have been carried out. A series of experiments are carried out to optimize the conditions of RNA isolation from pea plants. The established protocols will be used for subsequent experiments. The ultimate goal is to use the expressed sequence tags to dissect the infection process of pea by S. sclerotiorum at the molecular level. This information will be useful for develop resistance and control strategies for Sclerotinia white mold of pea and other plants.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Washington State University, Department of Plant Pathology. Additional details of research can be found in the report for the parent CRIS 5348-21000-014-00D, Germplasm Enhancement, Genetics and Disease Management of Cool Season Food Legumes. The project is aimed at developing cDNA libraries for expressed sequence tags of the pea pathogen Sclerotinia sclerotiorum and interactions between the pathogen and the host. The initial efforts were at selecting the most appropriate and representative isolate for the study. Information about general genetic variation among isolates of the pathogen was gathered and a phylogenetic tree was obtained. The information in the phylogenetic tree is used to select a subset of isolates for molecular comparisons based on which an representative isolate will be selected for the ESTs study in this project. Host
genotype Lifter, a recently released cultivar, was selected as host plant for this study to generate ESTs during infection by S. sclerotiorum. A series of experiments are carried out to optimize the conditions of RNA isolates and cDNA library construction from S. sclerotiorum and from pea plants. The established protocols will be used for subsequent experiments. The ultimate goal is to use the expressed sequence tags to dissect the molecular process of pea infection by S. sclerotiorum. This molecular information will be useful for develop resistance and control strategies for Sclerotinia white mold of pea.
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