Interaction between Brassica yellows virus silencing suppressor P0 and plant SKP1 facilitates stability of P0 in vivo against degradation by proteasome and autophagy pathways

Summary P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F‐box‐like motif reported to be essential for interaction with S phase kinase‐associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0–SKP1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br). We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0Br, which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br. Genetic analyses indicated that the P0Br–SKP1 interaction is not directly required for silencing suppression activity of P0Br, but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br. Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.


Fig. S3 P0 Br -mediated degradation of AGO1 is blocked by E-64d inhibitor. 6Myc-tagged
AtAGO1 was transiently co-expressed by Agrobacterium-infiltration in Nicotiana benthamiana leaves together with 3FLAG-tagged P0 Br and its mutant LP in the presence of P38 TCV . Total protein was extracted from co-infiltrated patches at 2 dpi. 50 μM E-64d was infiltrated into N.
benthamiana leaves for 12 h before harvesting (+) and DMSO treatment was used as a solvent control (-). Accumulation of 6Myc-tagged and 3FLAG-tagged proteins were analyzed by western blotting with c-Myc monoclonal antibody (α c-Myc) and FLAG monoclonal antibody (α FLAG), respectively. Coomassie stain of total proteins is shown to indicate equal loading (Coom.).

Fig. S4
Detection of Myc-tagged P0 Br and its mutants in yeast. Total proteins were extracted from yeasts. Accumulation of myc-tagged proteins were detected by western blotting with c-Myc monoclonal antibody (α c-Myc). Coomassie stains of total proteins are shown to indicate equal loading (Coom.).
Estradiol (100 μM) was applied to the non-transgenic (NT) or XVE:P0  benthamiana to induce expression of P0 Br -6Myc. Leaves were harvested 2 days after estradiol treatment for protein and RNA extraction. Accumulation of 6Myc-tagged P0 proteins were analyzed by western blotting with c-Myc monoclonal antibody (α c-Myc). Coomassie stains of total proteins are shown to indicate equal loading (Coom.).
Photographs were taken under long-wave length UV light at 2 dpi.

Fig. S7
Complementation analysis of P0 Br and its mutants. The mutant BrYV FS was coinfiltreated with 3FLAG-tagged P0 Br or its derivative mutants Q2A, LP, L184A, Y61D, Δ224-249, and Δ225-249. Empty vector (EV) co-infiltreated with BrYV FS or BrYV were used as negative and positive control, respectively. Accumulation of the mutant BrYV FS in Nicotiana benthamiana plants was analyzed by northern blotting. Total RNA and protein extracted from inoculated leaves at 2 dpi. Viral RNAs of BrYV were hybridized with a random primed 3′ UTR specific probe. Methylene blue staining of ribosomal RNAs after northern transfer was used as loading control for high molecular-weight RNAs blots (rRNA). The bands corresponding to viral genome RNAs (gRNA) and subgenomic RNAs (sgRNA) are indicated respectively at the right side of the panel. 3FLAG-tagged P0 Br and its derivative mutants were detected with FLAG monoclonal antibody (α FLAG). Coomassie stain of total proteins is shown to indicate equal loading (Coom.). TctcgagATGCAATTTGTAGC pGD-P0 Br -3FLAG cloning BrP0ApaR CgggcccTACAAACATTTCGG BrP0GGGApaR ATgggcccTCCTCCGCCTACAAACATTTCGG pGD-P0 Br -GFP cloning