Skip to main content
. 2021 May 13;12:2780. doi: 10.1038/s41467-021-23013-2

Fig. 1. The geminivirus-encoded C3 protein interacts with POLA2, the regulatory subunit of DNA polymerase α, which is required for geminiviral replication.

Fig. 1

a Schematic representation of bait (C3) and prey (SlPOLA2) isolated from the Y2H screen. Amino acid residues are indicated. SID: selected interaction domain. b C3 and POLA2 interact in yeast. AD: activation domain; BD: binding domain. The interaction between the SV40 large T antigen (T) and the tumor suppressor p53 is a positive control; empty AD- and BD-containing vectors (AD and BD, respectively) are used as a negative control. c C3-GFP (from TYLCV, BCTV, and TGMV) co-immunoprecipitates SlPOLA2-RFP (left) and NbPOLA2 (right) upon transient expression in N. benthamiana. IP: immunoprecipitate; IB: immunoblot; CBB: Coomassie brilliant blue. The predicted protein sizes are as follows: SlPOLA2-RFP, ~100 kDa; NbPOLA2-RFP, ~100 kDa; C3 (TYLCV)-GFP, ~42 kDa; C3 (BCTV)-GFP, ~42 kDa; C3 (TGMV)-GFP, ~42 kDa; GFP, ~26 kDa. Full blots and membranes can be found in the Source data file. d SlPOLA2 and NbPOLA2 interact with C3 from TYLCV, BCTV, and TGMV in BiFC assays upon transient expression in N. benthamiana. Fibrillarin-RFP marks the nucleolus and the Cajal body. Images were taken at 2 days post inoculation. Scale bar: 5 µm. Negative controls are shown in Supplementary Fig. 1. e Nuclear distribution of transiently expressed SlPOLA2-GFP, NbPOLA2-GFP, and free GFP in the absence (empty vector, EV) or presence of TYLCV in N. benthamiana. Scale bar: 5 µm. Additional images are shown in Supplementary Fig. 2. f NbPOLA2 binds the TYLCV genome in ChIP assays. The location of primers used for different genomic regions is shown in Supplementary Fig. 3a; the results for additional genomic regions are shown in Supplementary Fig. 3b. Data are the mean of three independent biological replicates; error bars indicate SD. ITS is used as the normalizer. g–i Viral accumulation in local (g, i; 3 days post inoculation) or systemic (h; 2 weeks post inoculation) TYLCV infections in POLA2-silenced (TRV-NbPOLA2) (g, h), POLA1-silenced (TRV-NbPOLA1) (i) or control (TRV) N. benthamiana plants measured by qPCR. Plants inoculated with the empty vector (EV) are used as a negative control. Data are the mean of six independent biological replicates; error bars represent SD. The 25S ribosomal DNA interspacer (ITS) was used as a reference gene; values are presented relative to ITS. The phenotype of silenced plants and silencing efficiency are presented in Supplementary Fig. 4. All experiments were repeated at least three times with similar results, with the exception of the ChIP assays, which were repeated twice. Asterisks indicate a statistically significant difference according to a two-sided Student’s t test (***P < 0.001; **P < 0.01). The original data from all experiments and replicates can be found in the Source data file.