Fig 1.
Average number of virus-encoded proteins and their molecular weight.
(A) Average numbers of virus-encoded proteins (per virus) in animal and plant viruses. (B) Molecular weight of viral proteins from animal and plant viruses. Sequences in (A and B) were downloaded from NCBI Virus, from complete RefSeq genome sequences of viruses infecting Viridiplantae (green plants, taxid: 33090) or Metazoa (metazoans, taxid: 33208).
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.
Fig 3.
CP is required and sufficient to change the subnuclear localization of C2.
(A) Subcellular localization of the TYLCV-encoded proteins fused to GFP at their C-terminus expressed alone (+EV; co-transformed with an empty vector control) or in the context of the viral infection (+TYLCV; co-transformed with a TYLCV infectious clone) in N. benthamiana leaves at 2 days post infiltration (dpi). Scale bar = 10 μm. EV: empty vector. (B) Subcellular localization of C2-GFP co-expressed with each of the viral proteins fused to RFP in N. benthamiana leaves at 2 dpi. Scale bar = 10 μm. AF: Autofluorescence. (C) Subcellular localization of C2-GFP or GFP-C2 when expressed alone (+EV) or co-expressed with CP (+CP) in N. benthamiana leaves at 2 dpi. The accumulation of the CP transcript is shown in S6A Fig. Scale bar = 10 μm. EV: empty vector. (D) Subcellular localization of C2-GFP when expressed alone (+EV) or in the context of the infection by the WT TYLCV virus (+TYLCV) or mutated versions unable to produce CP (+TYLCV-CPmut1; +TYLCV-CPmut2) in N. benthamiana leaves at 2 dpi. Scale bar = 10 μm. EV: empty vector. Viral accumulation is shown in S6B Fig. For details on TYLCV-CPmut1 and TYLCV-CPmut2, see Materials and Methods. In (B-D), the dashed circles mark the nucleolus. See also S6 Fig.
Fig 4.
C2 and CP functionally interact in planta and modify the transcriptome of N. benthamiana in an interdependent manner.
(A) Number of differentially expressed genes (DEGs) upon expression of C2, CP, or C2+CP in N. benthamiana leaves. UR: up-regulated; DR: down-regulated; ND: not detected; EV: empty vector. Full lists can be found in S2 Table. (B) Venn diagram of DEGs upon expression of C2 or C2+CP in N. benthamiana. UR: up-regulated; DR: down-regulated; EV: empty vector. (C) Heatmap with hierarchical clustering from samples in (A). The colour scale indicates the Z-score. EV: empty vector. (D) Functional enrichment analysis of up-regulated (UR) or down-regulated (DR) genes in the indicated samples. Gene Ontology (GO) categories from the Biological Process ontology enriched with a p-value<0.01 (up to top 10) are shown; functional enrichment was performed using the orthologues in Arabidopsis thaliana. “C2+CP vs. EV (only)” denotes the subset of genes that are down-regulated in this sample only, and not in the samples expressing the viral proteins separately. The colour scale indicates the -log10 (p-value), showing the significance of GO term enrichment. EV: empty vector. For a full list, see S3 Table. (E) Venn diagram of the GO terms (Biological Process ontology) over-represented in the subsets of down-regulated genes (p-value<0.01) in the different samples. DR: down-regulated; EV: empty vector. For a full list, see S3 Table. See also S7 Fig; S2 and S3 Tables.
Fig 5.
Expression of selected DEGs upon transient expression of C2, C2-GFP, or GFP-C2 in the presence and absence of CP in N. benthamiana leaves.
Gene expression was measured by RT-qPCR. The samples expressing CP or empty vector (EV) are used as control. Expression values are the mean of at least three biological replicates. Error bars represent SD. Asterisks indicate a statistically significant difference (*: p<0.05, **: p<0.01) according to a two-tailed comparison t-test. NbACT2 was used as the normalizer.
Fig 6.
C2 and CP functionally interact in planta in the context of the viral infection.
(A, B) Number of differentially expressed genes (DEGs) upon infection by TYLCV WT or C2-null or CP-null mutant variants (TYLCV-C2mut and TYLCV-CPmut1, respectively) in N. benthamiana leaves compared to the empty vector control (A), or to TYLCV WT (B). UR: up-regulated; DR: down-regulated; EV: empty vector. Full lists can be found in S2 Table. (C) Venn diagrams of DEGs upon infection by TYLCV C2-null and TYLCV CP-null mutants (TYLCV-C2mut and TYLCV-CPmut1, respectively) compared to TYLCV WT. UR: up-regulated; DR: down-regulated. (D) Heatmap with hierarchical clustering from (A). The colour scale indicates the Z-score. (E) Functional enrichment analysis of the subsets of up-regulated (UR) or down-regulated (DR) genes in the indicated samples. Gene Ontology (GO) categories from the Biological Process ontology enriched with a p-value<0.01 (up to top 10) are shown; functional enrichment was performed using the orthologues in A. thaliana. The colour scale indicates the -log10 (p-value), showing the significance of GO term enrichment. For a full list, see S4 Table. See also S8 and S9 Figs; S2 and S4 Tables.