Recombineering-based Gene Tagging in Arabidopsis

One of the main challenges of the post-genome era is deciphering the function of the over 30,000 genes encoded by the Arabidopsis genome. Translational fusions between the genes of interest (in their complete genomic context) and tags such as GFP provide the most reliable and detailed information of in vivo gene expression, a key step for understanding gene function. Classical molecular genetic approaches involving in vitro manipulation of large genomic DNA fragments are extremely labor-intense and unsuitable for whole-genome scale projects. Therefore, it would be highly desirable to develop alternative high-throughput methods to generate new tools that would enable the plant community to easily obtain accurate information of in vivo gene expression and protein localization for any given Arabidopsis gene.;Recent advances in E. coli-based homologous recombination systems (recombineering) allow for the rapid and precise modification of artificial chromosomes (BACs) without the need for restriction enzymes and classical cloning techniques. In Arabidopsis, where homologous recombination is highly inefficient, gene modifications in their chromosomal context can be achieved by recombineering of transformation-ready BAC clones (TACs). Using this system, tags can be precisely fused to the gene of choice in any given position, and subsequently reintroduced into the Arabidopsis genome by Agrobacterium-mediated transformation.;In this study, we proposed a pipeline suitable for recombineering-based gene tagging in Arabidopsis: 1) transformation of target gene containing TACs from JAtY clone to recombineering strain SW102, 2) two-steps recombination with galK positive/negative selection, 3) transformation of engineered TACs into Agrobacterium and 4) transforming Arabidopsis plants to examine expression pattern of a gene and the corresponding protein subcellular localization. 41 genes were selected to evaluate the efficiency and fidelity of each step in the pipeline as well as some of the factors that may affects such parameters. Our results indicate that tags can be efficiently and precisely inserted to a desired location in the gene of interests (GOI) and the modified TAC can be easily transformed into plant. Although deletions of these large T-DNAs are not uncommon during their integration in the plant genome, it is relatively easy to identify transgenic plants carrying a complete copy of the T-DNA. The practical utility of the system has been experimentally demonstrated and the expression pattern and protein localization of several genes examined. Importantly, this system was successfully used to make all the sequence modifications required to observe low-expressed and short-live proteins such as those belonging to the Arabidopsis Aux/IAA gene family. Tagged Aux/IAA proteins were proved to be nuclear-localized and trangenes were shown to display similar response to auxin treatment as endogenous genes. Finally, this procedure was optimized by: 1) eliminating the need for the low efficient second recombination step (replacement of the selectable marker by the fluorescent protein) using instead a much more efficient flipase-dependent step, 2) by removing bottle necks such as the preparation of competent cells for each individual clone using instead a pooling strategy, and 3) by developing a web tool to facilitate oligo design and clone selection. These optimizations were critical to quickly generate the constructs for 24 Aux/IAAs and setting the bases for a future whole genome tagging strategy.;Our results have demonstrated that the recombineering-based system can be adopted as a robust, efficient and reliable strategy for the generation of whole-gene reporter fusion constructs. This work has built a solid foundation for development of 96-well format recombineering pipeline to generate genome-wide gene-tag collection and provide detailed map of the Arabidopsis localizome.