2009 Annual Report
1a.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; and
4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources.
1b.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 (see attached letter of collaboration). 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 SCA with Oregon State University.
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