GAL4 transactivation-based assay for the detection of selective intercellular protein movement

Abstract

Several plant proteins function as intercellular messenger to specify cell fate and coordinate plant development. Such intercellular communication can be achieved by direct, selective, or nonselective (diffusion- based) traffi cking through plasmodesmata (PD), the symplasmic membrane-lined nanochannels adjoining two cells. A trichome rescue traffi cking assay was reported to allow the detection of protein movement in Arabidopsis leaf tissue using transgenic gene expression. Here, we provide a protocol to dissect the mode of intercellular protein movement in Arabidopsis root. This assay system involves a root ground tissuespecifi c GAL4/UAS transactivation expression system in combination with fl uorescent reporter proteins. In this system, mCherry, a red fl uorescent protein, can move cell to cell via diffusion, while mCherry-H2B is tightly cell autonomous. Thus, a protein fused to mCherry-H2B that can move out from the site of synthesis likely contains a selective traffi cking signal to impart a cell-to-cell gain-of-traffi cking function to the cell-autonomous mCherry-H2B. This approach can be adapted to investigate the cell-to-cell traffi cking properties of any protein of interest.