Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literaturecurated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.protein arrays | interactome | hormone | systems biology |
Arabidopsis thalianaA major objective in the postgenomic era is to assign detailed molecular function(s) to the many protein-coding genes that remain uncharacterized even in model organisms such as the reference plant Arabidopsis thaliana (1). It is estimated that Arabidopsis contains more than 2,000 transcription factors and transcriptional regulators (hereafter "TFs"), most of which are uncharacterized (2). TFs function as key players in plant hormone signal transduction pathways and are responsible for directing the widespread changes in gene expression that are essential for the regulation of growth and development in plants (3, 4). The TFs within these transcriptional regulatory networks do not work independently but rather undergo complex interactions with other proteins (2). Understanding how these TFs interact with other proteins will ultimately lead to a greater comprehension of biological systems.For determination of physical protein-protein interactions (PPIs), protein microarrays (5-10) are complementary to other PPI technologies such as the yeast two-hybrid system (Y2H) (11) and protein complex purification coupled with mass spectrometry (AP-MS) (12, 13). With conventional protein microarrays it is necessary to purify thousands of in vivo expressed proteins and to spot these purified proteins on a solid surface (5-8). In contrast, in situ synthesis protein microarray technologies simplify protein microarray fabrication by circumventing the steps of in vivo protein expression and purification (9,10,14,15). This streamlining facilitates an increase in the number of target genes that can be assayed, allowing for thousands of protein-encoding plasmids to be spotted at lower cost and in less time. Such ...