A functional genetic assay for nuclear trafficking in plants

Summary 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, NbImp α 1 and NbImp α 2 , 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 NbImp α ‐ 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.