Fig. 2: NIb is the principal viral factor inhibiting Pelota SUMOylation.

a In vivo SUMOylation of Pelota. Pelota-YFP and Myc-SUMO1 were co-expressed with Flag-tagged TuMV-encoded proteins in N. benthamiana leaves, and Pelota was immunoprecipitated with anti-GFP beads at 60 hpi. Immunoprecipitated proteins were then analyzed with anti-Myc, anti-Flag and anti-GFP antibodies. Stars indicate the Flag-tagged GUS and viral encoded proteins (P1, P3, CI, VPg, NIa, NIb, and CP). This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. b Pelota-YFP and Myc-SUMO1 were co-expressed with a gradient amount of NIb in N. benthamiana leaves. Note: samples were treated with MG132 for 6 h before collection to exclude the variations from protein degradation, and proteins were immunoprecipitated with anti-GFP beads at 60 hpi. Immunoprecipitated and input proteins were then analyzed with anti-Myc, anti-Flag, and anti-GFP antibodies. CBB-staining of RbcL was a loading control. Quantification of protein levels was performed using ImageJ software. Independent experiments were performed three times with similar results. The samples were derived from the same experiment and that blots were processed in parallel. c Growth phenotype of Col-0 and P3-OE #4 transgenic Arabidopsis plants. d qRT-PCR analysis of P3 RNA accumulation in P3-OE #4 transgenic Arabidopsis protoplasts. GFP, GFP-Pelota or GFP-Pelota with GFP-NIb were co-transformed in P3-OE #4 Arabidopsis protoplasts. The error bars indicate mean ± SD (n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Dunnett testing, **p = 0.0091, ns means not significant (p = 0.1030). e qRT-PCR analysis of P3 RNA accumulation at 40 hpi during expression of GUS and Pelota in N. benthamiana leaves transfected with buffer and NIb. The error bars indicate mean ± SD (n = 3 biologically independent samples with averaged technical duplicates shown), and **p = 0.0001, ns means not significant (p = 0.8688) (p values determined by two-tailed Student’s t test). f Virus symptoms and GFP fluorescence in TuMV-GFP infected plants after inoculation with GUS (mock), Pelota, Pelota+NIb. Plants were photographed under ambient or UV light at 8 dpi. g Analysis of TuMV-GFP RNA accumulations indicated in (f), systemically infected leaves. The error bars indicate mean ± SD (n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Dunnett testing, **p = 0.0020, ns means not significant (p = 0.9743).