2012 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, Universtiy of TN, Plant Pathologist, will provide the mutants using his TILLING resource funded through the National Science Foundation (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.
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 that show promise in the first experiments as a novel tool for silencing genes. 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. This research was conducted in support of objective 2A of the parent project.