Deliverables

Three deliverables will be offered to the research community:

Expression vectors harboring full-length sequences for each gene under its native promoter and tagged with YFP flanked by unique restriction sites,

2) Arabidopsis transgenic lines expressing each construct, and

3) A database describing the predicted structure of each protein, its expression pattern and intracellular localization. Also, a Reference Protein Subcellular Localization Map will be constructed using fluorescently-tagged proteins with known intracellular targeting.

Deliverable 1: Expression constructs harboring full-length genomic sequences for each gene under its native promoter and with the YFP tag flanked by two unique restriction sites engineered inside one of the exons. The tagged constructs will be supplied as smaller, transient expression TOPO vectors and as larger binary vectors for stable plant transformation. In addition to expressing the YFP-tagged genes, these vectors will be useful for other applications. For example, the YFP tag can be easily removed using its flanking unique restriction sites and replaced with other tags, such as T7 or HA, for immunodetection, or tags for fluorescence or bioluminescence resonance energy transfer (FRET/BRET) protein-protein interaction studies.

A similar CFP (Cyan fluorescent protein) tag with the flanking restriction sites will be engineered into a series of selected genes encoding proteins with known localization patterns for use as a standard control set for subcellular targeting,. Having confirmed the sequence of the clones, we will promptly list them on the TAIR website, and they will be immediately available as DNA stocks from ABRC. All vector DNA stocks will be supplied together with the description of how they were produced (restriction map, primers used for PCR, etc.) and instructions for their utilization (Agrobacterium transformation protocols, etc.).

Deliverable 2: Arabidopsis T-DNA insertion seed stocks of plant lines expressing the YFP- or CFP- tagged genes. After the expression of the tagged proteins has been confirmed, these seed stocks will be listed on the TAIR website and disseminated through ABRC.

Deliverable 3: A website and a searchable database containing information about the lines and constructs, including the gene sequences highlighted with the positions of primers and tagging sites, vector construct information, images and text descriptions of the protein expression pattern and intracellular localization, and protocols and standard operation procedures in experimentation, analysis, and interpretation. This database will be supplemented with the Reference Protein Subcellular Localization Map, generated using fluorescently-tagged proteins of known subcellular localization, and offered to other researchers to use as controls for studies of protein targeting. This information will include both the primary data, e.g., images of the cells and tissues, and our interpretations. It will be disseminated via unrestricted access to the project website integrated into The Arabidopsis Information Resource (TAIR). This database will leverage from the database schema and software developed at TAIR and also serve as (i) a module for integrating protein localization data and software to the existing data and software at TAIR, and (ii) an essential training data set for refining bioinformatic algorithms to better predict intercellular localization signatures for Arabidopsis and other organisms