Location: Horticultural Crops Research2010 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
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.