title: 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) trafficking through plasmodesmata (PD), the symplasmic membrane-lined nanochannels adjoining two cells. A trichome rescue trafficking 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 tissue-specific GAL4/UAS transactivation expression system in combination with fluorescent reporter proteins. In this system, mCherry, a red fluorescent 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 trafficking signal to impart a cell-to-cell gain-of-trafficking function to the cell-autonomous mCherry-H2B. This approach can be adapted to investigate the cell-to-cell trafficking properties of any protein of interest.
authors.affiliation: Division of Applied Life Science (BK21plus/WCU program), Plant Molecular Biology & Biotechnology Research Center, Gyeongsang National University, 27-306, 501 Jinju-Daero, Jinju, 660-701, Gyeong-Nam, Korea.
authors.forename: Dhinesh
authors.initials: D
authors.lastname: Kumar
authors.forename: Huan
authors.initials: H
authors.lastname: Chen
authors.forename: Yeonggil
authors.initials: Y
authors.lastname: Rim
authors.forename: Jae-Yean
authors.initials: JY
authors.lastname: Kim
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D031425:Plasmodesmata / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D019076:Transgenes; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein