Fig. 5: The SIM3 motif in NIb is required for interacting with SCE1.

a Predicted SUMO interacting motifs (SIMs) on the NIb protein. Replacement of the Valine (V), Isoleucine (I), and Leucine (L) with the Alanine (A) amino acid resulted in SIM mutations. b Identification of the interaction between SCE1 and the NIb (sim mutants) in yeast. Yeast cells co-expressing BD-SCE1 and AD-EV were used as negative control. c MBP-SCE1 protein combined with GST-Pelota was incubated with Flag-NIb (or sim mutants) in vitro. The proteins were immunoprecipitated with Amylose Resin. Input and pull-down proteins were analyzed with anti-GST, Flag, and MBP antibodies. This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. d BiFC assay showing interactions between Pelota, NIb, NIb^sim2 and NIb^sim3 in RFP-H2B (a nuclear marker, the red fluorescent protein fused to histone 2B) transgenic N. benthamiana leaves. Scale bars correspond to 20 μm. This experiment was repeated three times showing similar results. e Competitive Co-IP assays in vivo. SCE1-YFP and Myc-Pelota were co-expressed with NIb (or sim mutants)-Flag 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. The 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. f In vivo SUMOylation assay. Pelota-YFP and Myc-SUMO1 were co-expressed with NIb (or sim mutants)-Flag in N. benthamiana leaves. The proteins were immunoprecipitated with anti-GFP beads at 40 hpi and analyzed with anti-Myc, anti-Flag and anti-GFP antibodies. This experiment was repeated three times showing similar results. The samples were derived from the same experiment and that blots were processed in parallel. g Phenotypes of N. benthamiana plants infected by TuMV, TuMV-NIb^sim1, TuMV-NIb^sim2 and TuMV-NIb^sim3 at 10 dpi. Bar = 5 cm. h Phenotypes of B. napus plants infected by TuMV, TuMV-NIb^sim1, TuMV-NIb^sim2 and TuMV-NIb^sim3 at 15 dpi. Bar = 5 cm. i qRT-PCR analysis of TuMV, TuMV-NIb^sim1, TuMV-NIb^sim2 and TuMV-NIb^sim3 genomic RNA accumulation in (g, h). TuMV-GFP-NIb^sim2 genomic RNA accumulation was normalized to 1. The error bars indicate mean ± SD (n = 3 biologically independent samples with averaged technical duplicates shown). p values determined by one-way ANOVA and Tukey testing, ***p < 0.0001 (TuMV or TuMV-NIb^sim1 vs TuMV-NIb^sim2 or TuMV-NIb^sim3). ND means not detected.