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A functional genetic assay for nuclear trafficking in plants.
- Source :
-
The Plant journal : for cell and molecular biology [Plant J] 2007 Apr; Vol. 50 (1), pp. 149-58. Date of Electronic Publication: 2007 Mar 05. - Publication Year :
- 2007
-
Abstract
- The receptor importin-alpha mediates the nuclear import of functionally diverse cargo proteins that contain arginine/lysine-rich nuclear localization signals (NLSs). Functional homologs of importin-alpha have been characterized in a wide range of species including yeast, human and plants. However, the differential cargo selectivity of plant importin-alpha homologs has not been established. To advance nuclear import studies conducted in plant cells, we have developed a method that allows importin-alpha-dependent nuclear import to be assayed in Nicotiana benthamiana. We employed virus-induced gene silencing (VIGS) to knock down the expression of two importin-alpha homologs, NbImpalpha1 and NbImpalpha2, 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-alpha-dependent nuclear import of Nuk6 and Nuk7. In contrast, the nuclear import of Nuk12 and AtFib1 was unaffected in cells of NbImpalpha-silenced plants. These data suggest that P. infestans Nuk6 and Nuk7 proteins are dependent on one or more alpha-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.
- Subjects :
- Active Transport, Cell Nucleus
Amino Acid Sequence
DNA, Complementary chemistry
DNA, Complementary genetics
Gene Expression Regulation, Plant
Gene Silencing
Luminescent Proteins genetics
Luminescent Proteins metabolism
Microscopy, Confocal
Molecular Sequence Data
Nuclear Localization Signals genetics
Nuclear Localization Signals physiology
Phylogeny
Plant Proteins genetics
Plant Proteins metabolism
Protein Isoforms genetics
Protein Isoforms metabolism
Protein Isoforms physiology
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Reverse Transcriptase Polymerase Chain Reaction
Sequence Analysis, DNA
Nicotiana genetics
alpha Karyopherins genetics
alpha Karyopherins metabolism
Cell Nucleus metabolism
Plant Proteins physiology
Nicotiana metabolism
alpha Karyopherins physiology
Subjects
Details
- Language :
- English
- ISSN :
- 0960-7412
- Volume :
- 50
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- The Plant journal : for cell and molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 17346267
- Full Text :
- https://doi.org/10.1111/j.1365-313X.2007.03029.x