Location: Horticultural Crops Research2009 Annual Report
1a. 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
1b. 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.
3. Progress Report
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.