Progress 03/01/08 to 01/14/13

Outputs
Progress Report Objectives (from AD-416): 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416): Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. Documents Reimbusable with CSREES. Log 33891. This research was conducted in support of NP303 objective 2A of the parent project. Based on the previous results on P. sojae miRNAs, an endogenous miR gene discovered in Phytophthora sojae was used to construct a set of artificial miRNA constructs for transformation into P. sojae protoplasts. These artificial miRNA constructs contained the entire predicted miR gene foldback as well as the surrounding sequence, including predicted promoter elements. The only bases that were changed were the internal 18 bases in the miR and miR*, allowing directed targeting of genes of interest. In the initial analysis, the effector Avr1k, as well as DCL1 and DCL2, were targeted, and transformants were maintained under selection. Fewer DCL2-targeted transformants were produced than the other targets, which was consistent across multiple experiments. Transformants were analyzed for virulence on Glycine max, although the results were not conclusive. New target constructs were produced targeting GFP for a negative control and HISG to easily screen for auxotrophic transformants on media lacking supplemental histidine. Measurement of colony growth indicated several independent transformants with reduced growth on minimal media that increased upon addition of histidine or a full complement of amino acids. Of note is the apparent absence of transformed DNA signal in some of the affected transformants, indicating the potential for the artificial miRNA construct to induce silencing without permanently inserting into the genome. Further testing of the transformants is underway, including analysis of expression levels of the targeted genes as well as probing for the artificial miR oligos. DCL1 and DCL2 transformants that are found to have reduced expression of the target gene will be further analyzed for overall levels of small RNA production. This should allow differentiation between the two Dicer homologs as to which is involved in the 21- and 25/26- nucleotide small RNA production pathways. Artificial miRNA transformants are also being analyzed for efficiency of silencing and stability of that silencing by testing new transformants and tracking their target gene expression, then comparing these results to transformants that have been cultured over several months. This may be an effective alternative method to dsRNA transformation, which tends to have a very short-lived effect on target gene expression. Artificial miRNAs may also allow targeting of multiple genes with one construct, based on the nature of the short sequence used for targeting. One paper on the initial small RNA work, including the Phytophthora infestans Illumina HTS data, is nearly completed and ready for submission. Another paper focusing on the DCL and RDR homologs and their evolution within Stramenopiles is also soon to be completed, followed soon thereafter by a paper analyzing the evolution of the AGO homologs as well as the gene expression of all the small RNA biogenesis genes. A technical paper will be written to summarize the artificial miRNA data, and will likely be in collaboration with UC Riverside, which is working with the artificial miRNA construct in P. infestans. A final paper will summarize the results of the Illumina HTS data from P. sojae. To date, miRNA pathways have only been described for animals and plants and are newly characterized in the Stramenopile branch of the tree of life. By using the P. sojae miR RNA as a template to construct novel miR RNAs that can silence endogenous P. sojae genes, we have established the functionality of the miR RNA, and have also created a novel tool for genetic manipulation of P. sojae and other oomycetes.

Impacts
(N/A)

Publications

Progress 10/01/11 to 09/30/12

Outputs
Progress Report Objectives (from AD-416): 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416): Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. This research was conducted in support of objective 2A of the parent project. 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.

Impacts
(N/A)

Publications

Progress 10/01/10 to 09/30/11

Outputs
Progress Report Objectives (from AD-416) 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416) Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. 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 cllass 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.

Impacts
(N/A)

Publications

Progress 10/01/09 to 09/30/10

Outputs
Progress Report Objectives (from AD-416) 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416) Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. Documents Reimbusable with CSREES. Log 33891. 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 using either 454 or Illumina technologies. Small RNA sequences were mapped to their respective genomes. 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 biogenesis genes 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. Two distinct dicers, DCL1 and DCL2, were cloned from Phytophthora, and their gene structure and evolutionary relationships to other known dicers were described. Eight candidate miRNA genes from one gene family were identified in P. sojae, P. ramorum, and P. infestans. miRNA and small RNA derived from inverted repeats tend to belong to the 20-22nt size class while most other features tend to generate small RNA from the 24-26nt size class. Replicated small RNA libraries from three lifestages of P. sojae � mycelium, zoospores, and germinated cysts � were produced and sequenced. Analysis of this data, including discovery of potential miRNAs, is in progress. The identification of two predicted DCL proteins (DCL1 and DCL2) 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. We are in the process of functionally validating DCL1 and DCL2 function using several approaches including DCL mutants and miRNA silencing. The eight candidate miRNA genes provide the first evidence for presence of miRNA pathways in the oomycetes. To date, miRNA pathways have only been described for animals, plants and fungi and are newly characterized in the Stramenopile branch of the tree of life.

Impacts
(N/A)

Publications

Progress 10/01/08 to 09/30/09

Outputs
Progress Report Objectives (from AD-416) 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416) Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. Documents Reimbusable with CSREES. Log 33891. 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

Progress 10/01/07 to 09/30/08

Outputs
Progress Report Objectives (from AD-416) 1. To conduct a genome-wide expression profiling of small RNAs to identify sets of small RNAs specifically expressed during germination of cysts, growth in broth-shake culture and during infection of roots. 2. Locate and characterize the genome-wide distribution of small RNA- generating loci 3. Characterize the effect of DCR, RDR and AGO mutations on small RNA populations. 4. Development of a Phytophthora small RNA database and a public website and integration with other Phytophthora genome resources Approach (from AD-416) Our project will start with 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 roots by deep sequencing on the Illumina platform. Next we will complement small RNA analysis using tilling mutants in the key enzymes DCR and RDR which are responsible for synthesis of small RNAs. Select mutants and selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, the existing preliminary Phytophthora small RNA Database will be expanded, updated and improved. This database will provide a repository for sequences of small RNAs cloned from various Phytophthora spp., genotypes and tissues. The database will integrate tools to assist in miRNA and siRNA identification and analysis. Documents Reimbusable with CSREES. Log 33891. 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. Methods of ADODR monitoring included meetings, e-mail or other types of written correspondence.

Impacts
(N/A)

Publications