TY - JOUR
T1 - A functional genetic assay for nuclear trafficking in plants
AU - Kanneganti, Thirumala Devi
AU - Bai, Xiaodong
AU - Tsai, Chi Wei
AU - Win, Joe
AU - Meulia, Tea
AU - Goodin, Michael
AU - Kamoun, Sophien
AU - Hogenhout, Saskia A.
PY - 2007/4
Y1 - 2007/4
N2 - 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.
AB - 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.
KW - Agro-infiltration
KW - Importin-α
KW - Nuclear import
KW - Plant pathogen
KW - VIGS
UR - http://www.scopus.com/inward/record.url?scp=33947700386&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33947700386&partnerID=8YFLogxK
U2 - 10.1111/j.1365-313X.2007.03029.x
DO - 10.1111/j.1365-313X.2007.03029.x
M3 - Article
C2 - 17346267
AN - SCOPUS:33947700386
SN - 0960-7412
VL - 50
SP - 149
EP - 158
JO - Plant Journal
JF - Plant Journal
IS - 1
ER -