A functional genetic assay for nuclear trafficking in plants.
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.
Kanneganti
Thirumala-Devi
TD
Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH 44691, USA.
Bai
Xiaodong
X
Tsai
Chi-Wei
CW
Win
Joe
J
Meulia
Tea
T
Goodin
Michael
M
Kamoun
Sophien
S
Hogenhout
Saskia A
SA
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D010940:Plant Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins