Fig. 6: Potyvirid NIb-SIM2/3 is evolutionarily conserved and mediates interaction with SCE1.

a Comprehensive analysis of NIb sequences from 12 genera in the Potyviridae family, featuring a phylogenetic tree, multi-alignment of conserved sites, and a conservation heatmap. The tree branches depict the genetic divergence, with branch lengths indicating the degree of sequence variation. The multi-alignment of conserved sites showcases three SIM sites across the viruses. The right bubble illustrates the conservation levels of these sites across these virus sequences, with color intensity and bubble size representing the degree of conservation. b BiFC assay showing interactions between SCE1 and PVY, PVMV, and ANRSV NIb (as well as NIb^sim mutants) 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. c Analysis of the interactions between SCE1 and PVY, PVMV, and ANRSV NIb (as well as NIb^sim mutants) in yeast. d In vivo SUMOylation of Pelota. Pelota-YFP and Myc-SUMO1 were co-expressed with PVY, PVMV, and ANRSV NIb (as well as sim mutants) in N. benthamiana leaves, and Pelota was immunoprecipitated with anti-GFP beads at 40 hpi. Immunoprecipitated proteins were then analyzed with anti-Myc 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. e qRT-PCR analysis of P3 RNA accumulation at 40 hpi during expression of GUS or Pelota with PVY, PVMV, and ANRSV NIb (as well as NIb^sim mutants) in N. benthamiana 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 < 0.001.