Fig. 5

NbUBR7 modulates N protein level and functions as a negative regulator of N-mediated defense. a NbUBR7 overexpression reduced the stability of N in a proteasome-dependent manner. Agrobacterium containing expression sets consisting of an empty vector control (vector) or the agrobacterium containing the expression cassette of NbUBR7-HA or NbUBR7-MYC were infiltrated into N. benthamiana leaves followed by infiltration of MYC-tagged gN 24 later. 36 h after infiltration of gN, 50 µM proteasome inhibitor MG132 (+MG132) or the equal concentration of DMSO (−MG132) were further infiltrated into the pre-infiltrated leaves. Infiltrated leaf tissues were collected 12 h after DMSO or MG132 treatment and analyzed by western blot analysis. Antibodies used for the western blot analysis were indicated on the right of each panel. Equal protein loading is assessed by the similar amounts of PEPC protein. Molecular weight size markers in kDa are indicated on the left. Two independent samples (n = 2) for each treatment were analyzed in parallel. Band intensity was measured by Image J software and normalized to the PEPC protein control. Numbers below the top panel indicate the relative quantification of the corresponding band intensity, of which the empty vector control group was set to 100% [“±” indicates standard deviation (SD) of the mean]. b Double stranded hairpin-mediated silencing of NbUBR7 (NbUBR7-RNAi) enhances the stability of N. Empty vector control (vector) or the agrobacterium containing the hairpin NbUBR7 were infiltrated into the N. benthamiana leaves followed by infiltration of MYC-tagged N at 24 hpi. 36 h later, DMSO (-MG132) or MG132 (MG132) was then infiltrated into the pre-infiltrated leaves as described above. Infiltrated leaf tissues were collected 12 h after DMSO or MG132 treatment and analyzed by western blot. Three independent replicates (n = 3) were carried out for each treatment. Numbers below the top panel indicate the relative quantification of the corresponding band intensity, of which the MG132-treated empty vector control was set to 100% (“±” indicates SD). Equal protein loading is assessed by the similar amounts of PEPC protein (middle panel). Molecular weight size markers in kDa are indicated on the left. The downregulation of NbUBR7 in the infiltrated leaves were also confirmed by RT-qPCR (bottom panel). Data from three biological replicates were combined and values are shown as mean ± SD. c RNAi of NbUBR7 enhances the N-mediated resistance to TMV. Different regions of the leaf of N-containing transgenic N. benthamiana were first agroinfiltrated with hairpin NbUBR7 (1) and the control empty vector (2), respectively. 24 h later, TMV-U1-GFP was agroinfiltrated into the previously infiltrated regions. Photographs were taken under UV light at 5 dpi and representative results are shown (left panel). Leaf samples from the region 1 and 2 were then harvested and subjected to western blot analysis using the antibodies against GFP or PEPC (top right panels). RT-qPCR was performed to confirm the downregulation of NbUBR7 in the infiltrated region of the leaves (bottom right panel). Data from three biological replicates were combined and values are shown as mean ± SD. d Overexpression of NbUBR7 inhibits p50 effector-induced HR-PCD. Different leaf regions were infiltrated with MYC-tagged N (gN-6xMYC), TagCFP-tagged p50 (tCFP-p50) together with HA-tagged NbUBR7 (NbUBR7-HA) or citrine (Citrine-HA) control. Photographs were taken at 6 dpi and representative results are shown (left panel). The expression of citrine-HA or NbUBR7-HA was confirmed by Western blot analysis using anti-HA antibody (right panel). Arrowheads indicate the specific band of different HA fusions. Source data are provided as a Source Data file