BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation
Fig 5
Overexpression of POB1 reduces PUB17 protein accumulation in nuclei of N. benthamiana.
A. Western blot analysis indicates that GFP-POB1 overexpression destabilized HA-PUB17 in planta. HA- and GFP- fusions of PUB17 and POB1, respectively, along with GFP-only controls were expressed in N. benthamiana leaves using Agrobacterium-mediated transient assays. Total protein extracted after 3 days were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. Size markers (kD) are shown to the left of each immunoblot. B. POB1 localizes in the nucleus. Images are confocal projections and the close up nuclear confocal images (inset) shows the nucleoplasmic localisation of GFP-POB1. N. benthamiana leaves were examined by confocal microscopy 48 h after infiltration of 35S-GFP-POB1 constructs. The scale bar is 20 μm. C PUB17 was destabilized by POB1. Transient expression of YC-PUB17 alone, or co-expressed with YN-POB1 in N. benthamiana leaves, with or without treatment with MG132. Immunoblots with an anti-HA antibody showing stable protein fusions of YC-PUB17 and the anti-myc antibody shows stable protein fusions of YN-POB1 of the expected size. The lower panels show Ponceau staining (PS) of the membrane as a loading control. D. POB1 interacts with PUB17 in the nucleus. Confocal image of bimolecular fluorescence complementation (BiFC) following co-expression of YN-POB1 and YC-PUB17 in the presence of MG132 reveals YFP fluorescence in the nucleoplasm. The scale bar is 20 μm.