EMBO/soda-nel huggingface dataset:
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 1

Figure 1. BSMV infection disrupts chloroplast antioxidant defenses.

Chl-HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N. benthamiana epidermal cells. Ratiometric images (F488/405 nm) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast-targeted HyPer2, respectively. The false-blue color indicates the chloroplasts. Scale bars, 30 μm.

Quantification of the BSMV-dependent changes in Chl-HyPer2 fluorescence at 0 to 6 dpi. The percentage of ROS-positive chloroplasts (white) among all chloroplasts (blue and white) per visual field.

Western blot analysis of 2-Cys Prx redox status in empty vector (EV) and BSMV inoculated leaves under non-reducing conditions at 3 dpi. Western blots were analyzed with anti-2-Cys Prx or anti-CP antibodies. The Rubisco large subunit (RbcL) served as a loading control.

Relative mRNA levels of the sAPX, 2-Cys Prx, GPXL, and PrxQ genes in response to BSMV infections at 1, 3 and 5 dpi. The empty vector (EV) was used as a negative control, NbEF1α was used as an internal control.

BSMV accumulation in N. benthamiana leaves transiently expressing chloroplast ROS scavenging proteins. Agrobacterium harboring plasmids expressing various proteins are indicated above the panels. BSMV was agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blot analyses were conducted with anti-TGB1 or anti-Flag antibodies. RbcL served as the loading control. 2-CP, 2-Cys Prx.

BSMV accumulation in 2-Cys Prx or GPXL-silenced N. benthamiana plants using anti-TGB1 antibody. Empty vector TRV: 00 was used as negative control.

Data information: In (A), data are representative of at least three independent experiments. In (B), error bars indicate mean ± SEM (n = 5). a, b and c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05). In (C, E, and F), representative data are shown, at least three independent replicates were performed with similar results. In (D), error bars indicate mean ± SEM from three independent experiments; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 from Student's t test.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 2

Figure 2. BSMV γb interacts directly with NbNTRC in vivo and in vitro.

Y2H analyses of interactions between γb and NbNTRC. Various Y2H combinations are indicated on the images. Self-interactions of γb served as a positive control, and combinations containing either empty AD or BD constructs served as negative controls.

BiFC analyses of γb and NbNTRC interactions during BSMV infections. Various BiFC combinations are indicated on the left. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. Scale bars, 20 μm.

Co-IP analyses of interactions between γb and NbNTRC in N. benthamiana. Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Total and immunoprecipitated (IP) proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.

GST pull-down assays to verify interactions between γb and NbNTRC in vitro. GST-tagged NTRC or GFP were incubated with γb-His or GFP-His. Input and pull-down products were analyzed by Western blot analyses with anti-GST or anti-His antibodies. GFP-His and GST-GFP served as negative controls.

Co-localization of γb and NbNTRC during BSMV infection. NbNTRC-mCherry was co-expressed with BSMVγb-GFP (Jiang et al., 2020) in N. benthamiana, and confocal analyses were conducted at 3 dpi, and chloroplast autofluorescence is displayed as a false-blue color. Scale bars, 20 μm.

Data information: In (C and D), representative data are shown, and three replicates had similar results.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 3

Figure 3. His-85 of γb is required for NbNTRC interactions.

Y2H assay to analyze interactions between NbNTRC and γb mutants. Various Y2H combinations are indicated on the images. Interactions of γb and NbNTRC were confirmed earlier and served as a positive control, combinations containing an empty BD construct served as a negative control.

BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N. benthamiana. Various BiFC combinations are indicated at the top. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. RbcL-fused YFPc served as a negative control. Scale bars, 20 μm.

Co-IP analysis of interactions between NbNTRC and γb mutants. NbNTRC-3xFlag proteins together with γb-GFP, γbH85A-GFP or γbH85C-GFP were co-expressed in N. benthamiana leaves and harvested at 3 dpi. Total proteins were IP with anti-Flag beads. Total and IP products were detected in Western blots with anti-GFP or anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).

GST pull-down assays to evaluate in vitro interactions between NbNTRC and γb mutants. Purified γb-His, γbH85A-His, γbH85C-His, or GFP-His proteins were incubated with GST-tagged NbNTRC. Input and pull-down products were analyzed by Western blotting with anti-GST or anti-His antibodies. GFP-His served as a negative control. Arrowhead indicates the specific band of the target protein.

Analysis of VSR activities of γb mutants. Infiltrated N. benthamiana leaves co-expressing sGFP plus γbH85A-3xFlag, γbH85C-3xFlag or the pGD empty vector (EV) were observed at 5 dpi under long-wave UV illumination for GFP fluorescence. The γb-3xFlag protein served as a positive control. The white dashed circles indicate the infiltrated zone.

Western blot analysis of the GFP protein in co-infiltrated leaves at 5 dpi with anti-GFP antibody. RbcL was used as loading control.

Data information: In (C-F), representative data were shown, and three independent experiments showed a similar result.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 4

Figure 4. NTRC negatively regulates BSMV infection.

Effects of NbNTRC overexpression on BSMV protein accumulation. N. benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel. After 1.5 dpi, BSMV was inoculated onto the leaves by agroinfiltration. Three days later, total protein was extracted for Western blotting with anti-TGB1 and anti-Flag antibodies. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4). RbcL was a loading control.

Effects of NbNTRC overexpression on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel. NbNTRC and viral γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).

RT-qPCR analyses of viral RNA accumulation in 4B. NbPP2A was used as an internal control.

The symptoms of infection after BSMV inoculation of a different N. benthamiana plant at 8 dpi. Magnified images of leaves (in white boxes) are shown in the lower panels. Scale bars, 2 cm.

Statistical analysis of symptom appearance in different N. benthamiana plants after inoculation with BSMV.

Western blot analysis of the virus accumulation in Figure 4D with anti-TGB1 antibody. RbcL served as loading control.

Data information: In (A, B and F), representative data are shown and three eplicates similar results. In (C), error bars indicate mean ± SEM from three independent experiments. a and b indicate statistically significant differences among different groups (ANOVA, p-value < 0.05). In (E), error bars indicate mean ± SD from three independent experiments.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 5

Figure 5. The γb protein subverts NTRC-mediated antioxidant defenses.

Left panel: systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV γb mutants. Photographs were taken at 7 dpi. Right panel: schematic representation of inoculated (ino.), older systemic leaves (o-SL), and younger systemic leaves (y-SL) in N. benthamiana plants. Scale bars, 2 cm.

Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N. benthamiana leaves. Anti-TGB1 and anti-CP were used for protein detection.

Western blot detection of BSMV with anti-CP antibodies in upper N. benthamiana leaves at 7 days (o-SL) or 28 days (y-SL) after infiltration. o-SL, older systemic leaves; y-SL, younger systemic leaves.

Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants. A. tumefaciens harboring the indicated plasmids were infiltrated into N. benthamiana leaves. Note: Agrobacterium containing NbNTRC constructs were diluted 5-fold compared to normal use (OD600 = 0.3). BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blots were conducted with anti-TGB1 and anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).

Left panel: Western blot detection of BSMV CP accumulation in N. benthamiana leaves inoculated with BSMV and γb movement deficient mutants. Leaf samples were collected from different regions of the same leaf (right panel).

Effects of γb and γb mutants on 2-Cys Prx in N. benthamiana. Different combinantions of the pGD empty vector (EV) or γb and γb mutants are indicated above the panel. NbNTRC and γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies under reducing conditions, Western blot analyses of 2-Cys Prx abundance with anti-2-Cys Prx antibody under non-reducing conditions. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows, respectively.

Data information: In (B-F), representative data are shown, and three replicates had similar results. (B-E) RbcL served as loading control.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 6

Figure 6. γb competitively interferes with interactions between NTRC and 2-Cys Prx.

BiFC assays to identify interactions between NbNTRC and 2-Cys Prx in N. benthamiana during BSMV infection. NbNTRC-YFPc and 2-Cys Prx-YFPn were co-expressed with BSMV and γb mutant derivatives as indicated on the left. The EV was used as negative control. Scale bars, 30 μm.

Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software. The NbNTRC-2-Cys Prx interaction was normalized to that of the EV control, which was set to 1.0.

Split-LUC assays to determine NbNTRC-2-Cys Prx interactions in N. benthamiana during BSMV infection. NbNTRC-cLUC and 2-Cys Prx-nLUC were co-expressed in N. benthamiana leaves infected with the indicated BSMV derivatives, Bioluminescence was observed at 3 dpi. The white dashed circles indicate the infiltrated regions.

Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software.

Competitive GST pull-down assays showing the effects of γb on interactions between NbNTRC and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot analysis was performed using anti-GST and anti-His antibodies.

Analysis of NbNTRC-2-Cys Prx interactions in the presence of the γb protein. NbNTRC-3xFlag and 2-Cys Prx-GFP were co-expressed in N. benthamiana leaves. Total proteins were extracted at 4 dpi and immunoprecipitated with anti-Flag beads, co-incubated with increasing amounts (20, 40, and 60 μg) of γb-His and γb-His mutant derivatives. Input and IP proteins were analyzed by Western blotting with anti-His, anti-GFP or anti-Flag antibodies. The uncleaved 2-Cys Prx with cTP and cleaved 2-Cys Prx without cTP are marked with black and gray arrows, respectively.

Data information: Representative data were shown in (A and C), and three replicates had similar results. In B, error bars indicate the mean ± SEM, and a and b indicate statistically significant differences among different groups (n = 5, ANOVA, p-value < 0.05). In (D), error bars indicate mean ± SEM (n = 6). a, b and c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05). In (E, F), representative data are shown, and three independent experiments had similar results.

Source data are available online for this figure.
Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication (2021)
Figure 7

Figure 7. Interfering with antioxidant defenses may be a general strategy employed by chloroplast-replicating viruses

Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.

Quantification of LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.

Western blot analyses of 2-Cys Prx abundance in EV and LRSV inoculated leaves under non-reducing conditions at 3 dpi. RbcL served as a loading control.

Western blot analyses to detect LRSV γb in NbNTRC-OE and NbNTRC-KO N. benthamiana plants challenged with LRSV at 8 dpi. RbcL was used as the loading control.

Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.

Quantification of the TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.

Western blot analysis of 2-Cys Prx abundance in EV or TYMV inoculated leaves under non-reducing conditions at 5 dpi. RbcL was used as the loading control.

RT-qPCR analysis of TYMV RNA accumulation at 8 dpi. NbEF1α served as a internal control.

Data information: In (A and E), data are representative of at least three independent experiments. In (B and F), error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test. In (C, D, G, H), representative data are shown, and three replicates had similar results.

Source data are available online for this figure.
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 1

Fig 1. E2F7 and E2F8 are subjected to Cullin-RING ligase-dependent degradation during G2- and early M-phase.

Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.

Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.

Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.

Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 2

Fig 2. Cyclin F binds to E2F7 and E2F8 through a defined motif at the C-terminus.

Schematic view showing the location of putative cyclin F recognition motifs (RxL or RxI) on murine E2F7 and E2F8 proteins.

C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.

Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 3

Fig 3. E2F7 and E2F8 are targeted for ubiquitination and degradation by cyclin F during G2/M.

Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.

Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.

Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.

and E. Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 4

Fig 4. Failure to degrade E2F7 and E2F8 results in delayed G2/M progression.

Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.

Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.

Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.

Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.

Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.

Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).
Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression (2019)
Figure 5

Fig 5. Overexpression of E2F7R894A mutant delays G2/M progression

Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.

qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.

Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.