Comparative genomics provides a powerful window into ways that nature molds a genome over time. This project will rigorously evaluate the genetic and evolutionary basis of structure-functional relationships associated with different disease pathologies and conidiogenesis through comparative genome analyses of the close relatives Gaeumannomyces graminis var. tritici and Magnaporthe poae with the rice blast fungus Magnaporthe oryzae. G. graminis is the destructive agent of ‘take-all” disease of domesticated and wild grasses. It is also common on turf grasses and can be mistaken for summer patch caused by M. poae. Both fungi infect roots through the formation of a specialized structure called a hyphopodia. In contrast, M. oryzae, infects foliar tissue through the formation of an appressorium. The asexual spore states of these fungi also differ. Both M. poae and G. graminis have Phialophora anamorphs, whereas M. oryzae has a Pyricularia anamorph. The project objectives are:
1. to generate a 7X draft genome assemblies for G. graminis var. tritici and M. poae,
2. to generate ESTs from cDNA pools derived from G. graminis var. tritici and M. oryzae using 454 sequencing,
3. to provide a public database for each genome complete with automated gene predictions and annotations and
4. to create a user-friendly genome browser to enable phylogenomic analyses between G. graminis var. tritici, M. poae, and M. oryzae.
The broader impact of this work is that understanding genome-scale patterns of descent in these closely related taxa will reveal important clues to some fundamental recurrent issues in fungal biology, evolution and ecology, such as asexual growth, development and pathological adaptation. This project will provide opportunities for the Magnaporthe and Gaeumannomyces research communities to share their collective expertise and will provide meaningful research experiences for undergraduate students.
A High Throughput Protoplast System for Rice Functional Genomics and Proteomics: Protein-Protein Interactions at the Host-Pathogen Interface. NSF Plant Genome Program
One of the greatest challenges of modern molecular biology is to exploit the depth of genomic information generated over the past decade to understand the function of encoded genes within the context of an organism’s biology. Information to date is not sufficient to fully elucidate the function of these encoded genes. The interaction of proteins largely dictates form and function of living organisms, as well as the ability to perceive and respond to environmental signals, including potential pathogens. However, the ability of scientists to rapidly assess such protein-protein interactions remains a major challenge.
Rice is the food staple for over half the world’s population. Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is highly devastating and persists as a major threat to global food security. The ability of host and pathogen to detect and respond to each other’s presence is governed by protein interactions, however knowledge of these interactions remains fragmentary at best. Access to near complete genomes of both rice and M. grisea, along with the extensive biological resources and a rich history of investigation facilitated in part through prior NSF PGRP support, offers a relatively rare opportunity to investigate host-pathogen interactions and provides the motivation for this project.
The proposed research will develop and test a rice protoplast-based system to directly visualize protein interactions in living plant cells. The system will be developed by incorporating a novel screen based on the reassembly of dissected fragments of light/fluorescence generating proteins fused to associating fungal and/or rice peptides. As a proof-of-concept, the system will be first tested using rice proteins that are known to interact. The applicability of the system to interrogate the interaction of proteins from rice with those of the rice blast fungus, M. grisea, will be subsequently tested using a known avirulence protein and its cognate rice resistance receptor. The system will be finally scaled up to screen for unknown fungal and plant protein associations that we predict control the deployment of host defenses. The high throughput protoplast system will be also applied in a feasibility study to screen for unknown fungal elicitors of plant host defense responses, of which a select few will advance into the proposed fragment reassembly screen to identify interacting plant proteins.
The specific objectives are:
1. Development of a protoplast based system for high throughput screening of fungal proteins that elicit host defense responses.
2. Development of a marker fragment reassembly technique for direct visualization of protein-protein interactions in rice protoplasts.
3. Advance education and outreach programs stemming from proposed research.
This project was designed with specific attention to broad impacts on human resource enrichment and the development of technologies needed to advance the field of plant genomics/proteomics. Outreach and engagement opportunities are important to the success of this project, and as such, an extensive education plan for high school students and activities promoting recruitment and participation by members of underrepresented groups are planned. Significantly, the technologies to be developed will have far-reaching utility for the discovery and evaluation of other protein-protein or protein-ligand interactions in plant systems outside rice and rice blast.
Characterization of the transcription circuitry regulating pathogenicity in the rice blast fungus. USDA-CSREES NRI Functional Genomics Program
In this project, we propose to identify regulatory subnetworks of genes that control infection related development and pathogenicity using the rice blast system. Magnaporthe grisea is widely considered a seminal model for studying fungal parthenogenesis of plants. Significant progress has been made to define the central signaling networks, which include the cAMP, MAP kinase and Ca/calmodulin signal pathways, but we know little of the downstream targets and the genes they regulate. Whole genome microarray studies have revealed that several hundred genes are differentially expressed during appressorium formation and infection, but the transcriptional circuitry that regulates their expression remains a mystery. A common feature of these core pathways is that they activate downstream transcription factors through phosphorylation. First, we propose to identify transcription factor targets of pathogenesis associated kinases by performing phosphorylation assays using protein microarrays containing Magnaporthe transcription factors. Second, a subset of these transcription factors selected based on phosphorylation patterns, expression profiles, and other functional criteria will be used to identify the sets of genes they regulate. This will be accomplished by identifying the specific DNA (promoter sequences) in vivo to which these transcription factors bind using chromatin immunoprecipitation and DNA microarrays, commonly referred to as ChIP-chip. We plan to initiate our ChIP-chip studies with two transcription factors previously shown to regulate pathogenicity in Magnaporthe. Results from ChIP-chip and functional analyses (gene knock-out) will be integrated with existing differential gene expression data in order to assemble the underlying regulatory subnetworks. This work will contribute to enhancing protection and safety of the Nation’s agriculture and food supply (NRI and USDA Strategic Plan goal 3).
Specific objectives:
1. Refine the M. grisea genome annotation and identify all putative transcription factors.
2. Identify transcription factors that are targets for phosphorylation by MAPK, cAMP dependent, and calcium dependent protein kinases using protein chips.
3. Identify binding motifs and target genes of a selected set of transcription factors phosphorylation specificity.
4. Perform functional studies on TFs and the genes they regulate.
Gene ontology terms for standardized annotation of plant-associated microbe genomes. USDA-CSREES NRI/NSF Microbial Genome Program
Plant-associated microbes span diverse kingdoms of life, including bacteria, fungi, oomycetes, nematodes and viruses. These microbes have all evolved mechanisms to solve the same problem, namely how to evade, suppress or neutralize the defense systems of their plant hosts. However, our ability to identify these mechanistic similarities among diverse organisms is greatly impeded by the lack of a set of standard terms to describe how these microbes interact with plants. The overall goal of this proposal is to extend the Gene Ontology with terms describing molecular functions, biological processes and cellular structures used by bacteria, fungi, oomycetes and nematodes for establishing associations with plants.
The specific aims are:
1. As participants in the Gene Ontology (GO) Consortium, expand our development of new GO terms and relationships for products of genes implicated in plant-interactions in the bacteria Erwinia chrysanthemi, Pseudomonas syringae pv tomato and Agrobacterium (3 species), the fungus Magnaporthe grisea, the oomycetes Phytophthora sojae and Phytophthora ramorum, and the nematode Meloidogyne hapla.
2. Use the terms to annotate genes implicated directly in plant association by experimental evidence and also genes implicated by bioinformatic approaches such as sequence similarity and divergence rates. Use evidence codes to indicate the basis for annotation. The annotation process will drive the development of the lower level (i.e. more specific) terms.
3. Evaluate methods for automating the transfer of Plant-Associated Microbe (PAM) GO term annotations to genomes of other plant-associated microbes related to the seven lead species.
4. Create reference gene sets for the wider community to use in generating automated GO annotations that have improved quality and relevance for plant-associated microbes.
5. Carry out training and outreach activities to engage the wider microbial genomics community in the use of PAM GO terms for annotation, functional genomics, and education, including three annual training workshops.
The broader impact of this work will be to establish a means for greatly facilitating the exchange of information about plant-associated microbes from diverse kingdoms of life, across diverse database systems holding that information. This exchange of information will advance our knowledge of how microbes establish associations with plants, and hence facilitate the improvement of technologies for protecting plants from pathogens and promoting the role of beneficial microbes. Many of the terms will also be of value to researchers studying animal- and human-microbe interactions. Researchers in the community will be trained in the use of the ontologies and the PAMGO terms, via training workshops, internships, presentations at meetings, participation in term development via a listserve, and on-line training documents. Training these researchers will result in a concomitant advance in the utility of their own work back to the community, as they begin using GO terms. Training will emphasize graduate students, postdoctoral fellows and junior faculty, especially those who are minorities, are from undergraduate institutions and/or are from EPSCoR states. Researchers from overseas countries lacking established research funding infrastructures will also be encouraged to receive training. Project PIs will incorporate information about GO into their graduate and undergraduate courses.
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