Combinatorial interactions between viral proteins expand the potential functional landscape of the tomato yellow leaf curl virus proteome
Fig 2
The proteins encoded by the plant DNA virus tomato yellow leaf curl virus (TYLCV) associate with one another in the plant cell.
(A) Viral protein-protein interactions detected in yeast two-hybrid. The minimal synthetic defined (SD) medium without leucine (Leu), tryptophan (Trp), histidine (His), and adenine (Ade) was used to select positive interactions; SD without Leu and Trp was used to select co-transformants (S1 Fig). The interaction between the SV40 large T antigen (T) and the murine tumor suppressor p53 is a positive control. AD: activation domain; BD: binding domain. This experiment was repeated twice with similar results. (B) Summary of viral protein-protein interactions detected by co-immunoprecipitation (co-IP) in the absence (-) or presence (+) of TYLCV. These experiments were repeated at least three times; the colour scale represents the percentage of positive interaction results among all replicates, with 1 = 100%. The original co-IP blots are shown in S2 Fig (in the absence of TYLCV) and S3 Fig (in the presence of TYLCV). An interaction between two viral proteins was considered as positive if at least two replicates showed positive interactions either in the absence or presence of TYLCV. (C) Viral protein-protein interactions detected by bimolecular fluorescence complementation (BiFC) in N. benthamiana leaves. nYFP: N-terminal half of the YFP; cYFP: C-terminal half of the YFP. Images were taken at 2 days post-infiltration (dpi). Scale bar = 10 μm. This experiment was repeated at least four times; combination with Hoechst staining and negative controls can be found in S4 Fig. Additional images are shown in S5 Fig. (D) Viral protein-protein interactions detected by split-luciferase assay in N. benthamiana leaves. nLuc: N-terminal part of the luciferase protein; cLuc: C-terminal part of the luciferase protein. Images were taken at 2 dpi. The colour scale represents the intensity of the interaction in counts per second (CPS). This experiment was repeated three times with similar results. (E) Summary of the intra-viral protein-protein interactions identified in (A-D). Different colours represent different methods, as indicated; circle size indicates the number of the methods in which a positive interaction was detected. (F) Network of intra-viral protein-protein interactions. The colored lines indicate the positive interactions detected by Y2H, Co-IP, BiFC, split-luciferase assay, or AP-MS. See also S1–S5 Figs; S1 Table.