Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta

Plant J. 2009 Jul;59(1):150-62. doi: 10.1111/j.1365-313X.2009.03850.x. Epub 2009 Feb 26.


Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Gene Expression Regulation, Plant
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microscopy, Confocal
  • Nicotiana / genetics*
  • Nicotiana / metabolism
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Protein Transport
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Red Fluorescent Protein
  • Transformation, Genetic


  • Cyan Fluorescent Protein
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins