A functional genetic assay for nuclear trafficking in plants
The receptor importin-α mediates the nuclear import of functionally diverse cargo proteins that contain arginine/lysine-rich nuclear localization signals (NLSs). Functional homologs of importin-α have been characterized in a wide range of species including yeast, human and plants. However, the differential cargo selectivity of plant importin-α homologs has not been established. To advance nuclear import studies conducted in plant cells, we have developed a method that allows importin-α-dependent nuclear import to be assayed in Nicotiana benthamiana. We employed virus-induced gene silencing (VIGS) to knock down the expression of two importin-α homologs, NbImpα1 and NbImpα2, which we identified from N. benthamiana. Agro-infiltration was then used to transiently express the NLS-containing proteins Arabidopsis thaliana fibrillarin 1 (AtFib1) and the Nuk6, Nuk7 and Nuk12 candidate effector proteins of the oomycete plant pathogen Phytophthora infestans. In this manner, we demonstrate importin-α-dependent nuclear import of Nuk6 and Nuk7. In contrast, the nuclear import of Nuk12 and AtFib1 was unaffected in cells of NbImpα-silenced plants. These data suggest that P. infestans Nuk6 and Nuk7 proteins are dependent on one or more α-importins for nuclear import. Our VIGS-based assay represents a powerful new technique to study mechanisms underlying the transport of proteins from cytoplasm to nucleus in plants.
Document Type: Research Article
Affiliations: 1: Department of Entomology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA 2: Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA 3: Molecular and Cellular Imaging Center, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA, and 4: Department of Plant Pathology 201F Plant Science Building, University of Kentucky, Lexington, KY 40546, USA
Publication date: April 1, 2007