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Study of Molecular Basis for Pathogen Recognition and Signal Transduction Leading to Disease Resistance in Plants
EPA Grant Number: GF950776Title: Study of Molecular Basis for Pathogen Recognition and Signal Transduction Leading to Disease Resistance in Plants
Investigators: Holzberg, Steven Paul
Institution: University of California - Berkeley
EPA Project Officer: Broadway, Virginia
Project Period: September 1, 1995 through January 1, 2000
Project Amount: $26,394
RFA: STAR Graduate Fellowships (1995)
Research Category: Fellowship - Health , Academic Fellowships , Health Effects
Description:
Objective:The purpose of this research is to study the relationship between structure and function of the proteins encoded by the N gene in tobacco which has shown resistance to virus in order to enhance understanding of the mechanisms whereby plants actively resist disease. Plants are naturally capable of actively resisting infections by viral, bacterial, fungal and nematode pathogens through local responses at the infection site which prevent the spread of the invading organism. One of the most common mechanisms employed by the plant is the initiation of the hypersensitive response (HR), which is characterized by localized cell death in the area immediately surrounding the initial infection site. This study involves two stages: 1) localization of the N protein in the plant cell, and 2) identification and characterization of protein-protein interactions necessary for N function. In the first stage, determining the subcellular localization of the N protein is a fundamental step towards understanding its function. This study will produce a series of antibodies to N and use these for in situ localization and cellular fractionation experiments. The antibodies will be generated using full length and partial N proteins expressed in E. coli cells using one of several commercially available expression and purification systems. Once purified, these antigens will be used to produce monoclonal and polyclonal anti-sera. These anti-sera will then be used to investigate the cellular localization of the N gene products in the various types of tobacco cells at various points before, during and after infection with standard immunohistochemical techniques. In the second stage, protein-protein interactions are critical to the functions of most proteins and this has proven especially true for those involved in regulatory and signal transduction circuits. The initial focus will involve a protein-protein interaction trap known as the yeast two-hybrid system. If the proteins interact in a specific manner, they will be capable of activating transcription of an appropriate reporter gene. Once an interaction has been identified, the appropriate mutants will be constructed and tested to identify the specific structures involved. Any findings from the two-hybrid system, or any in vitro approach, will be confirmed in vivo, using transgenic tobacco plants as well as sequence analysis and genetic mapping, where appropriate. Finally, transgenic tobacco plants will be employed for in vivo analysis of the partner protein's functional relationship to N. Alternatively, the relevance of the partner protein can be addressed by both its over expression and under expression in separate transgenic plants.
Supplemental Keywords:Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, RFA, Ecosystem/Assessment/Indicators, Molecular Biology/Genetics, Biology, exploratory research environmental biology, Ecology, Ecological Risk Assessment, Ecological Indicators, Biochemistry, Environmental Microbiology, Environmental Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecosystem Protection, Ecology and Ecosystems, pathogen recognition, plants, tobacco plants, hypersensitive response (HR), transduction, signal transduction, disease resistance, transgenic plants, N gene, pathogens, disease resistant plant