2010 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.
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.
ADODR monitoring included site visits, lab meetings, e-mail and phone calls.