Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. The genome sequence of P. sojae and several other Phytophthora species has revealed that their genomes contain an enormous repertoire of genes potentially involved in infection. However, information about the regulatory mechanisms controlling expression of these genes is greatly lacking. Oomycete species are notorious for their genetic variability, rapidly adapting to overcome chemical controls and host genetic resistance. However the underlying mechanisms governing this variability are not at all understood. Small RNAs, including siRNAs and miRNAs, have been demonstrated to play a major role in modulating the expression of eukaryotic genomes. Oomycete genomes encode the machinery necessary to generate several classes of small RNAs, but there is absolutely no information about the roles that small RNAs play in oomycete biology and pathology. The goal of this proposal is to fill in this major gap in our understanding of this important group of pathogens. The specific aims of the proposal include: 1. To use genome-wide, high-throughput sequencing to identify all small RNA-generating loci expressed specifically in mycelia, during germination of cysts, and during infection of plants; 2. Analyze the genome-wide distribution of loci encoding all small RNA classes; 3. Characterize the effects of mutations in the P. sojae Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) genes on small RNA classes, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources. Approach (from AD-416): Our project will start with genome-wide small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean hypocotyls by deep sequencing using the Illumina 1G system. We will then identify and analyze TILLING mutants with defects in the DCL and RDR genes, which we propose catalyze biogenesis of the two P. sojae small RNA size classes. Collaborator, Universtiy of Tennessee, Plant Pathologist, will provide the mutants using his TILLING resource funded through the National Science Foundation (NSF). Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the Phytophthora small RNA database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs identified from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in small RNA identification and analysis. The comprehensive database will be publicly available through the current Phytophthora genome web interface at VBI and the small RNA database resources in the Carrington lab and CGRB at OSU. The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. This proposal focuses on genome-wide analysis of small RNAs expressed during different life stages and during infection of soybean by the pathogen Phytophthora sojae. Three small RNA libraries from the Phytophthora species P. infestans, P. sojae and P. ramorum were produced and sequenced using high throughput sequencing technologies. Two small RNA size classes, 21 and 25 nucleotides in size, were identified in each library. A database and genome viewer were developed to facilitate analysis of the small RNA component in Phytophthora. Putative RNA silencing biogenesis genes were tentatively identified in the P. sojae, P. infestans, and P. ramorum genomes: Two distinct dicer genes, named dicer-like 1 and dicer-like 2, were cloned from Phytophthora, and their gene structure and evolutionary relationships to other known dicers were described. The gene structure for another small RNA biogenesis effector, RNA-dependent RNA polymerase, was also determined. A special class of small RNAs termed micro RNAs was discovered. Eight candidate micro RNA genes from one gene family were identified in P. sojae, P. ramorum, and P. infestans. We are now evaluating using artificially designed micro RNAs as tools for targeted, stable silencing in Phytophthora that show promise in the first experiments as a novel tool for silencing genes. To date, micro RNA pathways have only been described for animals and plants and are newly characterized in the Stramenopile branch of the tree of life. Thus, this is the first report of presence of micro RNAs in a new branch of the tree of life. Artificial micro RNAs show promise as a novel tool for research and potentially management of Phytophthora. This research was conducted in support of objective 2A of the parent project.
Impacts
(N/A)
Publications
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. The genome sequence of P. sojae and several other Phytophthora species has revealed that their genomes contain an enormous repertoire of genes potentially involved in infection. However, information about the regulatory mechanisms controlling expression of these genes is greatly lacking. Oomycete species are notorious for their genetic variability, rapidly adapting to overcome chemical controls and host genetic resistance. However the underlying mechanisms governing this variability are not at all understood. Small RNAs, including siRNAs and miRNAs, have been demonstrated to play a major role in modulating the expression of eukaryotic genomes. Oomycete genomes encode the machinery necessary to generate several classes of small RNAs, but there is absolutely no information about the roles that small RNAs play in oomycete biology and pathology. The goal of this proposal is to fill in this major gap in our understanding of this important group of pathogens. The specific aims of the proposal include: 1. To use genome-wide, high-throughput sequencing to identify all small RNA-generating loci expressed specifically in mycelia, during germination of cysts, and during infection of plants; 2. Analyze the genome-wide distribution of loci encoding all small RNA classes; 3. Characterize the effects of mutations in the P. sojae Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) genes on small RNA classes, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources. Approach (from AD-416) Our project will start with genome-wide small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean hypocotyls by deep sequencing using the Illumina 1G system. We will then identify and analyze TILLING mutants with defects in the DCL and RDR genes, which we propose catalyze biogenesis of the two P. sojae small RNA size classes. Collaborator, Universtiy of TN, Plant Pathologist, will provide the mutants using his TILLING resource funded through the National Science Foundation (NSF). Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the Phytophthora small RNA database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs identified from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in small RNA identification and analysis. The comprehensive database will be publicly available through the current Phytophthora genome web interface at VBI and the small RNA database resources in the Carrington lab and CGRB at OSU. Documents Grant with Virginia Bioinformatics Institute. The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. This proposal focuses on genome-wide analysis of small RNAs expressed during different life stages and during infection of soybean by the pathogen Phytophthora sojae. Three small RNA libraries from the Phytophthora species P. infestans, P. sojae and P. ramorum were produced and sequenced using high throughput sequencing technologies. Two small RNA size classes, 21 and 25 nucleotides in size, were identified in each library. A database and genome viewer were developed to facilitate analysis of the small RNA component in Phytophthora. Putative RNA silencing biogenesis genes were tentatively identified in the P. sojae, P. infestans, and P. ramorum genomes: Two distinct dicer genes, named dicer-like 1 and dicer-like 2, were cloned from Phytophthora, and their gene structure and evolutionary relationships to other known dicers were described. The gene structure for another small RNA biogenesis effector, RNA-dependent RNA polymerase, was also determined. A special class of small RNAs termed micro RNAs was discovered. Eight candidate micro RNA genes from one gene family were identified in P. sojae, P. ramorum, and P. infestans. We are now evaluating using artificially designed micro RNAs as tools for targeted, stable silencing in Phytophthora. To date, micro RNA pathways have only been described for animals and plants and are newly characterized in the Stramenopile branch of the tree of life. Thus, this is the first report of presence of micro RNAs in a new branch of the tree of life. Artificial micro RNAs show promise as a novel tool for research and potentially management of Phytophthora. The project was monitored by meetings, e-mail, and phone calls.
Impacts
(N/A)
Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. The genome sequence of P. sojae and several other Phytophthora species has revealed that their genomes contain an enormous repertoire of genes potentially involved in infection. However, information about the regulatory mechanisms controlling expression of these genes is greatly lacking. Oomycete species are notorious for their genetic variability, rapidly adapting to overcome chemical controls and host genetic resistance. However the underlying mechanisms governing this variability are not at all understood. Small RNAs, including siRNAs and miRNAs, have been demonstrated to play a major role in modulating the expression of eukaryotic genomes. Oomycete genomes encode the machinery necessary to generate several classes of small RNAs, but there is absolutely no information about the roles that small RNAs play in oomycete biology and pathology. The goal of this proposal is to fill in this major gap in our understanding of this important group of pathogens. The specific aims of the proposal include: 1. To use genome-wide, high-throughput sequencing to identify all small RNA-generating loci expressed specifically in mycelia, during germination of cysts, and during infection of plants; 2. Analyze the genome-wide distribution of loci encoding all small RNA classes; 3. Characterize the effects of mutations in the P. sojae Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) genes on small RNA classes, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources. Approach (from AD-416) Our project will start with genome-wide small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean hypocotyls by deep sequencing using the Illumina 1G system. We will then identify and analyze TILLING mutants with defects in the DCL and RDR genes, which we propose catalyze biogenesis of the two P. sojae small RNA size classes. Collaborator Kurt Lamour will provide the mutants using his TILLING resource funded through NSF. Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the Phytophthora small RNA database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs identified from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in small RNA identification and analysis. The comprehensive database will be publicly available through the current Phytophthora genome web interface at VBI and the small RNA database resources in the Carrington lab and CGRB at OSU. Documents Grant with Virginia Bioinformatics Institute. We have continuously attempted to obtain isolates of P. ramorum from across the United States many of which have or are being genotyped. We continue to follow up on every new outbreak and have obtained isolates from current as well as historic outbreaks. We were also finally able to obtain isolates from the APHIS collection and genotyping is conducted on all isolates. We have been able to modify existing protocols for genotyping P. ramorum based on AFLP or SSR. Currently, we use a modified multiplexed protocol to identify genotypes and place them into one of three known clonal lineages. We have also adapted protocols for determining mating type that became necessary once we found the mixed A1/A2 infection in Humboldt County, CA. This effort confirmed mating types for this find. Applying our protocols all new finds for which we were able to obtain cultures were determined to belong to lineage NA1, NA 2, or EU1. In one study, we examined the genetic diversity of P. ramorum in U.S. nurseries by microsatellite genotyping 279 isolates collected from 19 states between 2004 and 2007. Of the three known P. ramorum clonal lineages, the most common lineage in the sample was NA1. The EU1 and NA2 clonal lineages had more limited distributions and lower genetic diversities. Migration pathways were revealed by a single genotype shared among the majority of states and in the clustering of NA1 isolates into only two groups, one containing isolates from Connecticut, Oregon, and Washington and the other isolates from California and the remaining states. At the same time, several states showed genetic diversities as high as the three West Coast states and two-thirds of multilocus genotypes were limited in their distribution to one state. Together, these data suggest that migration, rapid mutation, and genetic drift all play a role in structuring the genetic diversity of P. ramorum in U.S. nurseries. The inferred connections between states were consistent with USDA APHIS trace- forward and trace-back analyses revealing two predominant migration routes from the West coast originating either in California or the Pacific Northwest. This work demonstrates that analysis of variable microsatellites can be used to recreate the evolutionary history and putative migration patterns of clonal pathogens thus showing promise for similar forensic applications in other clonal organisms. A selection of information is regularly migrated to our website (http://oregonstate.edu/%7Egrunwaln/phytophthora.php) onto a searchable, relational database using MySQL. Methods of ADODR monitoring included meetings, e-mail or other types of written correspondance.
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. The genome sequence of P. sojae and several other Phytophthora species has revealed that their genomes contain an enormous repertoire of genes potentially involved in infection. However, information about the regulatory mechanisms controlling expression of these genes is greatly lacking. Oomycete species are notorious for their genetic variability, rapidly adapting to overcome chemical controls and host genetic resistance. However the underlying mechanisms governing this variability are not at all understood. Small RNAs, including siRNAs and miRNAs, have been demonstrated to play a major role in modulating the expression of eukaryotic genomes. Oomycete genomes encode the machinery necessary to generate several classes of small RNAs, but there is absolutely no information about the roles that small RNAs play in oomycete biology and pathology. The goal of this proposal is to fill in this major gap in our understanding of this important group of pathogens. The specific aims of the proposal include: 1. To use genome-wide, high-throughput sequencing to identify all small RNA-generating loci expressed specifically in mycelia, during germination of cysts, and during infection of plants; 2. Analyze the genome-wide distribution of loci encoding all small RNA classes; 3. Characterize the effects of mutations in the P. sojae Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) genes on small RNA classes, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources. Approach (from AD-416) Our project will start with genome-wide small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean hypocotyls by deep sequencing using the Illumina 1G system. We will then identify and analyze TILLING mutants with defects in the DCL and RDR genes, which we propose catalyze biogenesis of the two P. sojae small RNA size classes. Collaborator Kurt Lamour will provide the mutants using his TILLING resource funded through NSF. Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the Phytophthora small RNA database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs identified from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in small RNA identification and analysis. The comprehensive database will be publicly available through the current Phytophthora genome web interface at VBI and the small RNA database resources in the Carrington lab and CGRB at OSU. Documents Grant with Virginia Bioinformatics Institute. Significant Activities that Support Special Target Populations Phytophthora species and related oomycete pathogens cause tens of billions of dollars of damage each year to a huge range of agriculturally and ornamentally important plants. They also do severe damage to forests and threaten entire natural ecosystems. Because of their threat, several oomycetes are listed as bioterrorism agents. The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. Three small RNA libraries from P. infestans, P. sojae and P. ramorum were produced and sequenced. Two small RNA size classes, with peaks at 21 and 25 nucleotides, were identified in each library. In other species, multiple size classes are reflective of distinct biogenesis pathways. These peaks were identified in analyses of both total reads and unique sequences. A database and genome viewer were developed to facilitate analysis of the small RNA component in Phytophthora. Putative RNA silencing proteins were tentatively identified in the P. sojae, P. infestans, and P. ramorum genomes, with known, functional DCL, RDR, AGO from plants, animals, fungi and protists as query sequences. The identification of two predicted DCL proteins in Phytophthora immediately suggests that the two small RNA size classes identified in the small RNA libraries sequenced to date could be formed through distinct DCL functions. Cloning and sequencing of mRNA and DNA for DCL and RDR are in progress. This project addresses CSREES goal #3: To enhance protection and safety of the Nations agriculture and food supply under the category of �Fundamental research: Research testing scientific hypotheses and providing basic knowledge that enables advances in applied research and from which major conceptual breakthroughs are expected to occur.� It addresses the following program priority of 51.0 Microbial Genomics (B): Functional Genomics of Microorganisms: �1) Characterization of mechanisms of pathogenicity by microorganisms�. Methods of ADODR monitoring included meetings, e-mail or other types of written correspondence.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The overall goal of our research is to identify the genetic mechanisms that enable oomycete pathogens to overcome host defenses, using the soybean pathogen Phytophthora sojae as a model. The genome sequence of P. sojae and several other Phytophthora species has revealed that their genomes contain an enormous repertoire of genes potentially involved in infection. However, information about the regulatory mechanisms controlling expression of these genes is greatly lacking. Oomycete species are notorious for their genetic variability, rapidly adapting to overcome chemical controls and host genetic resistance. However the underlying mechanisms governing this variability are not at all understood. Small RNAs, including siRNAs and miRNAs, have been demonstrated to play a major role in modulating the expression of eukaryotic genomes. Oomycete genomes encode the machinery necessary to generate several classes of small RNAs, but there is absolutely no information about the roles that small RNAs play in oomycete biology and pathology. The goal of this proposal is to fill in this major gap in our understanding of this important group of pathogens. The specific aims of the proposal include: 1. To use genome-wide, high-throughput sequencing to identify all small RNA-generating loci expressed specifically in mycelia, during germination of cysts, and during infection of plants; 2. Analyze the genome-wide distribution of loci encoding all small RNA classes; 3. Characterize the effects of mutations in the P. sojae Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) genes on small RNA classes, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources. Approach (from AD-416) Our project will start with genome-wide small RNA analysis in P. sojae lifestages and infected roots, followed by functional analysis of P. sojae mutants. First, we will document the small RNA repertoire in pure P. sojae lifestages and in infected soybean hypocotyls by deep sequencing using the Illumina 1G system. We will then identify and analyze TILLING mutants with defects in the DCL and RDR genes, which we propose catalyze biogenesis of the two P. sojae small RNA size classes. Collaborator Kurt Lamour will provide the mutants using his TILLING resource funded through NSF. Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the Phytophthora small RNA database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs identified from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in small RNA identification and analysis. The comprehensive database will be publicly available through the current Phytophthora genome web interface at VBI and the small RNA database resources in the Carrington lab and CGRB at OSU. Documents Grant with Virginia Bioinformatics Institute. Significant Activities that Support Special Target Populations This project started July 3, 2008. There is no progress to report. Methods of ADODR monitoring include meetings, e-mail, and phone calls.
Impacts
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Publications
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