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Abstract, Phase 1 (completed)
Plants respond to pathogen attack through a variety of signaling pathways consisting of a large number of regulatory as well as effector genes. During the past several years, many defense-related genes have been identified through genetic analysis conducted in Arabidopsis thaliana. Importantly, Arabidopsis exhibits all of the major kinds of defense responses present in other plants. Although a relatively large number of Arabidopsis defense-related genes have been identified, progress in developing overall explanatory models of plant-pathogen interactions is currently limited by two major experimental roadblocks. First, most of the phenotypic tests that have been used to characterize pathogen-host interactions do not have sufficient discriminatory power to assign defense-related genes to specific signal response pathways. To circumvent this limitation, large-scale transcript profiling analyses will be carried out on appropriately selected Arabidopsis defense-related mutants, including double and triple mutants. Combining genetic epistasis analysis with genomic technologies should lead to the development of a much more detailed model of how the various defense-related genes function and interact in combating pathogen attack. The second factor limiting the understanding of the plant defense response is that it is simply not possible to analyze the overwhelming volume of current data using conventional methods. The large volume of microarray data that will be generated in the near future compounds this problem. Moreover, data from different laboratories are not directly comparable because standardized experimental conditions are not employed. To help mitigate the problems associated with the analysis of large data sets, a sophisticated web-accessible plant-microbe interaction database (PMIDB) will be created to provide a common repository and standardized format for experimental data. The specific aims of the project are to:
1) Use transcript-profiling
analysis to identify Arabidopsis defense-related genes and construct a
custom microarray (pathoarray) enriched for defense-related genes. These
custom pathoarrays will be made available to the Arabidopsis community
at a nominal cost with the expectation that the experimental results generated
using these pathoarrays will be deposited in PMIDB.
2) Use the pathoarrays
from Aim 1 and Arabidopsis defense-related mutants to define the expression
signatures resulting from the activation of defense pathways.
3) Create a web-accessible
plant-microbe interaction database (PMIDB). This database will be developed
during the next four years and will contain standardized experimental
procedures for analyzing host defense responses, a list of all the pathogenesis-related
mutants and their phenotypes, a list of defense-related genes with links
to various sequence databases, and expression profiles of different plant-pathogen
interactions and different defense-related mutants.
This 2010 Project
provides a unique opportunity for applying genomic approaches to genetic
analysis of plant defense mechanisms. Understanding the mechanisms of
plant defense is of interest not only to basic science but also to development
of agriculture and protection of the environment.
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Abstract, Phase 2
This project will determine the function of transcription factors (TFs) in the Arabidopsis defense-response signaling network (see current candidate list). TFs are key switches that control plant defense responses against pathogens, and expression profiling using gene chip (microarray) is the most powerful genomics approach available for high through-put analysis of transcriptional events. In Aim 1, profiling will identify candidate TFs in understudied defense pathways, including those induced by the so-called "PAMP" molecules which are commonly associated with pathogens of plants as well as animals. In Aim 2, mutants in the candidate TF genes (see current list) will be identified to see whether disrupting the genes and their functions will affect any defense response. In Aim 3, the TFs identified in Aim 2 will be linked to GFP, a protein that glows under UV, to mark and sort cells expressing these TFs, and profiling will be conducted in these cells to identify genes whose expression patterns mirror those of the TFs (i.e., cohort transcriptomes). The important TFs will also be modified so their transport into the nucleus where they function can be controlled experimentally and their target genes will be identified. Collectively, these experiments will provide spatial, temporal, and hierarchical information on defense-related transcriptional events, which is currently lacking but needed to build a model of the transcriptional network of interacting defense pathways. All data obtained in this project will be deposited in IMDS (Integrated Microarray Database System)
and made available to the public either at the time of publication or after a 6-month delay,
whichever comes sooner."
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