Abstract
Maize is a powerful model system for functional genomics in the grain crops, because its reference genome has recently been sequenced and functional tools are increasing. However, new methods of investigating gene function within cells and tissues are needed to understand protein functions at the cellular and sub-cellular level, which will ultimately provide guiding information for crop improvement. This project aims to generate tools in maize that can be used to drive expression of any gene in a number of specific chosen tissue or cell types. The use of these tools will permit the visualization of reporter genes with unprecedented spatial and temporal resolution, and will also enable functional gene over-expression or gene knockout studies. In addition, this project will make use of newly improved fluorescent protein color tags to label sub-cellular or developmental compartments, and will develop live imaging techniques for growing maize plants to demonstrate the utility of all of the reporter lines for functional studies. The project will therefore transform functional genetic experiments in maize, by providing a means to understand gene function in specific cells and tissues during development.
The project will deliver to the research community a permanent stock of stably transformed seeds for 50 tissue specific activator/reporter lines, 20 fluorescent-tagged protein lines, as well as fully characterized in vivo imaging methods. It will also develop a pipeline for handling large image datasets that will be applicable to other projects. All resources, including images, seeds and gene constructs, will be publicly available via this project website http://www.weizhongli-lab.org/cellgenomics. For long-term availability, images will be migrated to the community database, MaizeGDB, and seeds will be deposited with the Maize Genetics Stock Center.
Maize cells consist of interconnected but discrete compartments that help to maintain cellular function and order. Identifying proteins that localize to these compartments is critical to understanding developmental and physiological processes in maize, which in turn provides guiding information for crop improvement. This project makes use of recent advances in genomics to identify proteins that localize to diverse cellular compartments. The project will generate reporter lines that express proteins tagged with a fluorescent marker. Using confocal microscopy, the lines will display visual information about when and where the tagged proteins are expressed and how specific proteins may be interacting and functioning. Genes for tagging will provide complete marker coverage of cellular compartments. Several resources will be used for gene selection, including known genes from the auxin/cytokinin hormone pathway and from the RAB-mediated vesicle trafficking pathways. Gene selections will also be guided by public gene models generated from maize genome sequencing projects and by specific requests from the maize research community. The outcomes of the project will include a set of stable tagged lines expressing fluorescent protein-derived tags for 100 proteins, which will represent full coverage of cellular compartments.
The tagged gene constructs will be freely available from the Jackson lab, and seeds of transgenic lines will be available from the Maize Genetics Stock Center. Access to project outcomes: Data on characterization of the lines at the cellular level will be compiled in a localization catalog posted on this public website http://www.weizhongli-lab.org/cellgenomics. Cell biology workshops will help train the scientific user community in analysis of the tagged lines using confocal microscopy. The project will also serve tribal community colleges in the Rocky Mountain West by enhancing education in cell and molecular biology. The project will therefore generate resources that bridge cell biology and functional genomics and will provide training across diverse scientific and learning communities. Training and outreach activities will be reported on the Plant Genome Research Outreach Portal.