Source: USDA-ARS, HORTICULTURAL CROPS RESEARCH LAB submitted to
GENOME-WIDE FUNCTIONAL ANALYSIS OF PHYTOPHTHORA SMALL RNAS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
0212863
Grant No.
2008-35600-18780
Project No.
ORER-2007-04672
Proposal No.
2007-04672
Multistate No.
(N/A)
Program Code
51.0B
Project Start Date
Jan 15, 2008
Project End Date
Jan 14, 2013
Grant Year
2008
Project Director
Grunwald, N. J.
Recipient Organization
USDA-ARS, HORTICULTURAL CROPS RESEARCH LAB
3420 NW ORCHARD AVENUE
CORVALLIS,OR 97330
Performing Department
(N/A)
Non Technical Summary
Phytophthora species and related oomycete pathogens cause severe damage to a huge range of agriculturally and ornamentally important plants, and to forests and natural ecosystems. To develop improved methods for controlling Phytophthora infection, it is essential to understand the genetic and regulatory mechanisms controlling how these pathogens break down or evade plant defense and cause disease. Small RNAs have been demonstrated to play major roles in modulating the expression and maintenance of eukaryotic genomes. Oomycete genomes encode small RNA biogenesis and effector proteins and express at least two classes of small RNA, but there is no information about small RNA function in oomycete biology and pathology. By analogy with other eukaryotes such a smice or humans, it is expected that many genes involved in pathogenesis may be subject to transcriptional or posttranscriptional regulation that involves small RNAs. This proposal focuses on genome-wide analysis of small RNAs expressed during different life stages and during infection of soybean by P. sojae.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240991040100%
Goals / Objectives
This proposal focuses on genome-wide analysis of small RNAs expressed during different life stages and during infection of soybean by P. sojae. The specific aims 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 P. sojae small RNA populations, growth, morphology and pathology; 4. Develop a publicly accessible Phytophthora small RNA database that integrates with existing Phytophthora genome resources.
Project Methods
This 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 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. Selected mutants will be analyzed in the mycelial lifestage and in life stages in which a phenotype is expressed. Throughout, a 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.

Progress 01/15/09 to 01/14/10

Outputs
OUTPUTS: 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. PARTICIPANTS: Stephanie Bollmann, post-doctoral scientist (USDA ARS): cloned DCL1, DCL2, and RDR genes and described gene structure. Noah Fahlgren, PhD candidate (OSU): conducted bioinformatics analysis of 454 and Illumina sequence data patterns of small RNA expression in Phytophthora and identified two distinct size classes corresponding to DCL1 and DCL2. Emily Feldman, undergraduate researcher (Virginia Tech): conducted isolation of P. sojae RNA and screened TILLING mutant lines. Todd Brengel undergraduate researcher (Virginia Tech): conducted isolation of P. sojae RNA and screened TILLING mutant lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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.

Publications

  • - Haas, B. J. et al. 2009. Genome sequence and comparative analysis of the Irish potato famine pathogen Phytophthora infestans. Nature 461, 393-398.


Progress 01/15/08 to 01/14/09

Outputs
OUTPUTS: 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. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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.

Publications

  • No publications reported this period