Development of genomic resources using fluorescent proteins in maize

This project is funded by the NSF Plant Genome Program.

Recent advances in sequencing technology have generated genome sequences from nearly all groups of organisms. These massive datasets have revolutionized the types of biological questions that can now be addressed. Despite the ability to predict the function of some proteins by comparative bioinformatics, experimental validation remains necessary to assign function definitively. One effective approach to validate gene function is to develop visualization and biochemical tools associated with fluorescent proteins (FPs).

We have created a public resource of cellular biomarkers that we and other members of the maize community use for both small-scale analysis of individual genes as well as for global expression and functional profiling. To date, we have generated over 100 stable, natively expressed, maize FP fusion lines that highlight most sub-cellular compartments, and allow us to study various cellular pathways including hormone signaling, cytoskeletal behavior, vesicle trafficking and stem cell maintenance and differentiation. We are also using these lines to immunoprecipitate tagged proteins to identify previously uncharacterized components of these important pathways. In the case of FP-tagged transcriptional regulators, chromatin immunoprecipitation (ChIP) is being used to identify putative DNA binding sites and/or to confirm gene targets that have been detected by transcriptional profiling of the respective mutant. To study maize development on a broader scale, we are optimizing the LhG4 2-component trans-activation expression system for use in maize. This system relies on the transcription factor LhG4 being expressed by a cell or tissue specific promoter, to drive the expression of a reporter in trans, via its pOp binding sites. The reporter can be any gene, such as GUS, FPs, an FP fusion protein, a gene of interest for tissue specific expression, a silencing construct for transcriptional knockdowns, or a gene encoding a toxin for tissue ablation experiments. Data on the characterization of our lines, including confocal micrographs, movies, and recent publications can be found on our website, http://maize.jcvi.org/cellgenomics/index.php.