Fig 1.
NtPUB17 directly interacts with NtPOB1.
A. Yeast-2-Hybrid assays indicating that full length and ARM domain of NtACRE276 (NtPUB17) interact with NtPOB1. The various indicated proteins were expressed as AD- and BD- fusions in AH109 yeast cells. Transformed AH109 cells were grown on selective media (Leu-, Trp-, His- and X-α-Gal) to test for interacting partners and on nonselective media (Leu-, Trp-) to test for the transformation efficiency. BD-SV40 and AD-p53 fusion proteins were used as positive controls for interaction in yeast. B. Immunoprecipitation analysis indicates that GFP-NtPUB17 and HA-NtPOB1 interact in planta. HA- and GFP fusions of NtPOB1 and PUB17 respectively were expressed in tobacco leaves using Agrobacterium-mediated transient assays. Total protein was extracted after 3 days and incubated with anti-GFP agarose beads to immunoprecipitate GFP-PUB17. The protein extracts (input) as well as the immunoprecipitates (IP) were analyzed by western blot using anti-HA and anti-GFP antibodies.
Fig 2.
NtPOB1 is a negative regulator of avr9 –Cf9 mediated HR-PCD.
A. RT-PCR analysis of the NtPOB1 transcript levels in silenced and non-silenced tobacco plants 3 days after Agroinfiltrations with stated hairpin constructs. ACTIN transcript levels were used as loading controls. B. Leaf segments expressing vector only, HG: 00, or NtPOB1-silencing HG:NtPOB1 construct were infiltrated with Avr9 peptide 3 days after agroinfiltrations. Pictures were taken 36 hours after Avr9 treatment. At this time point NtPOB1-silenced leaf segments showed much earlier onset of HR-PCD than leaf segments expressing vector only. HR-PCD assays were repeated in at least three different leaves in three different Cf-9 tobacco plants. Representative leaf image is shown of HR-PCD that was observed in 80% (8/10) of the leaves tested.
Fig 3.
NbPOB1 is a negative regulator of basal defence and HR-PCD specified by diverse pathogen PAMPs and effectors.
A and B Growth of Ps pv. tabaci in NbPOB1-silenced plants expressing TRV:00 (empty vector) or TRV:NbPOB1_A (A), or TRV:NbPOB1_B (B). Bacterial counts were performed 3 and 5 days post-inoculation (dpi). C and D P infestans infection assays indicating lesion growth measurement (C) and sporangia spore counts (D) in plants expressing TRV:00, TRV:NbPOB1_A or TRV:NbPOB1_B. E. Graphs show the percentage of infiltration sites developing clear HR-PCD (Y-axis) at 4 dpi of the PAMP INF1, and R genes/cognate effectors on N. benthamiana plants expressing TRV:00, TRV:NbPOB1_A and TRV:POB1_B. Experiments were repeated at least three times, each with 3 leaves from no less than 4 dedicated plants and error bars indicate +/- SE, * represents a P<0.05 statistically different (ANOVA) from TRV:GFP controls (n = 84 inoculations for INF1 on each of TRV:00, TRV:NbPOB1_A or TRV:NbPOB1_B; n = 84 for Pto/AvrPto; n = 78 for Cf4/Avr4; and n = 72 for Rx/PVX CP). F Photographs of typical infiltration zones at 4 dpi.
Fig 4.
Over-expression of POB1 enhances P. infestans colonisation and suppresses HR-PCD.
A. Over-expression of N. benthamiana NbPOB1 suppresses the generation of the Avr9-elicited HR cell death in Cf-9 tobacco plants. Agrobacteria containing the 35S: GFP-NbPOB1 and 35S:GFP were inoculated into opposing tobacco leaf segments (marked with white dotted line). Avr9 peptide was infiltrated into the leaf segments 3 days after Agroinfiltrations. HR-PCD assays were repeated at least in three different leaves each in three different Cf-9 tobacco plants. Representative leaf image is shown that was observed in 70% (14/20) of the leaves tested. Pictures were taken 2 days after Avr9 treatment. B As in A, but expressing GFP-AtPOB1 fusion protein. Representative leaf image is shown that was observed in 70% (14/20) of the leaves tested. C Immunoblot analysis of NbPOB1and AtPOB1 in Cf-9 tobacco leaves. Total protein was extracted from leaves expressing GFP, GFP-NbPOB1 or GFP-AtPOB1, 3 days after Agroinfiltrations. Samples were immunoblotted with anti-GFP antibodies (top panel). Rubisco large subunit (rbcL) was used as a loading control as indicated by Ponceau staining (PS). D. Overexpression of GFP-POB1 in N.benthamiana enhances P. infestans leaf colonisation. Agroinfiltration was performed of either 35S: GFP-POB1 or 35S: GFP on opposing leaf segments of N. benthamiana. The leaves were then challenged with P. infestans 1 day after agro-infiltration. Quantification of lesion diameter 5dpi post inoculation. Statistical analysis was carried out using ANOVA with pairwise comparisons performed with a Holm-Sidak test; *** indicates p≤0.001 (n = 152); error bars show standard error. The graph represents the combined data from 4 biological replicates. E Graphs show the percentage of infiltration sites developing HR-PCD (Y-axis) at 4 days post-infiltration (dpi) of INF1 and R gene/cognate effector pairs, co-expressed with either 35S:GFP alone or 35S:GFP-POB1 on N. benthamiana plants. Experiments were repeated at least three times, each with no less than 8 dedicated plants (n = 132 inoculation sites for INF1; 108 for Rx/PVX; 124 for Cf4/Avr4; and 120 for Pto/AvrPto). Error bars indicate +/- SE and * represents a P<0.05 statistical difference (ANOVA) from GFP control. F Photographs of typical infiltration zones (white dotted lines) in E at 7 dpi.
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.
Fig 6.
POB1 suppression of plant immunity occurs in plant nuclei.
A. NES-GFP-POB1 was excluded from the nucleus. A representative N. benthamiana leaf expressing NES-GFP-POB1 examined by confocal microscopy 48 h after infiltration of NES-GFP-POB1, with close-up nuclear confocal image inset. The scale bar is 20 μm. B. GFP-POB1 and NES-GFP-POB1 fusion proteins are intact in planta. Immunoblots were probed with anti-GFP antibody. The lower panels show Ponceau staining of the membrane as a loading control (PS). C. GFP-POB1 significantly enhances P. infestans colonization (p≤0.001, n = 109), but NES-GFP-POB1 fails to do so. Mean lesion diameter of P. infestans colonisation following expression of GFP control, GFP-POB1 and NES-GFP-POB1.The lesion diameter was measured 5 days post inoculation. Statistical analysis was carried out using ANOVA with pairwise comparisons performed with a Holm-Sidak test. Error bars show standard error. The graph represents the combined data from 3 biological reps. D. Representative images of P. infestans colonisation in C. E. GFP-POB1 suppresses HR-PCD triggered by Cf4/Avr4, but NES-GFP-POB1 fails to suppress this (letters denote whether results are statistically significantly different, p≤0.001, n = 51). Mean % HR-PCD of Cf4/Avr4 co-expressed with GFP control, GFP-POB1 and NES-GFP-POB1 Statistical analysis was carried out using ANOVA with pairwise comparisons performed with a Holm-Sidak test; error bars show standard error. F. Representative images of HR-PCD in E.
Fig 7.
POB1 dimerisation is critical for cell death suppression and PUB17 turnover.
A. POB1 D146A mutant protein cannot dimerize in yeast. Wildtype POB1 and POB1D146A mutant proteins were expressed as AD- and BD- fusions in AH109 yeast cells. Transformed AH109 cells were grown on selective media (Leu-, Trp-, His- and X-α-Gal) to test for interacting partners and on nonselective media (Leu-, Trp-) to test for the transformation efficiency. BD-SV40 and AD-p53 fusion proteins were used as positive controls. B Immunoprecipitation analysis of the POB1D146A mutation on POB1 dimerization. HA- and GFP fusions of the wild-type POB1 and mutant POB1 D146A were expressed in N. benthamiana leaves. The protein extracts and immunoprecipitates (IP) were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. As indicated barely any HA-POB1D146A co-immunoprecipitates with the GFP-POB1D146A. C. Transient over-expression of GFP-POB1 and GFP-POB1D146A in Cf-9 tobacco. Agrobacteria containing the indicated constructs were infiltrated into Cf-9 tobacco leaf segments. Avr9 peptide was infiltrated into the leaf segments 3 days after agroinfiltrations. HR-PCD assays were repeated in at least three different leaves in three different Cf-9 tobacco plants. Representative leaf image is shown where HR developed in leaves (9/20) expressing detectable AtPOB1 D146A. Pictures were taken 2 days after Avr9 treatment. D. Graph of HR-PCD of Cf4/Avr4 co-expressed with GFP-POB1 or GFP-AtPOB1D146A. Statistical analysis was carried out using ANOVA with pairwise comparisons performed with a Holm-Sidak test; and letters denote whether results are statistically significantly different, p≤0.001 (n = 99); error bars show standard error. The graph represents the combined data from 4 biological reps. E. Immunoprecipitation analysis of the POB1D146A mutation on POB1 interaction with PUB17. GFP fusions of the wild-type POB1 and mutant POB1 D146A were expressed in N. benthamiana leaves along with HA-PUB17 and treated with proteasome inhibitor MG132. The protein extracts (input) and immunoprecipitates (IP) were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. As indicated barely any GFP-POB1D146A immunoprecipitatied with PUB17-HA.