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) (P0^Br). We found that several residues within P0^Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0^Br, 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 P0^Br. Genetic analyses indicated that the P0^Br–SKP1 interaction is not directly required for silencing suppression activity of P0^Br, but it facilitates stability of P0^Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0^Br. Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0^PL-IM) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0^PL-IM has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0^PL-IM F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0^PL-IM. As has been observed with other P0 proteins, P0^PL-IM suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0^PL-IM fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0^PL-IM may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

ORF3a, a newly identified non-AUG-initiated ORF encoded by members of genera Polerovirus and Luteovirus, is required for long-distance movement in plants. However, the mechanism of action of P3a in viral systemic movement is still not clear. In this study, sequencing of a brassica yellows virus (BrYV) mutant defective in systemic infection revealed two-nucleotide variation at positions 3406 and 3467 in the genome. Subsequent nucleotide substitution analysis proved that only the non-synonymous substitution (C→U) at position 3406, resulting in P3a^P18L, abolished the systemic infection of BrYV. Preliminary investigation showed that wild type BrYV was able to load into the petiole of the agroinfiltrated Nicotiana benthamiana leaves, whereas the mutant displayed very low efficiency. Further experiments revealed that the P3a and its mutant P3a^P18L localized to the Golgi apparatus and near plasmodesmata, as well as the endoplasmic reticulum. Both P3a and P3a^P18L were able to self-interact in vivo, however, the mutant P3a^P18L seemed to form more stable dimer than wild type. More interestingly, we confirmed firstly that the ectopic expression of P3a of other poleroviruses and luteoviruses, as well as co-infection with Pea enation mosaic virus 2 (PEMV 2), restored the ability of systemic movement of BrYV P3a defective mutant, indicating that the P3a is functionally conserved in poleroviruses and luteoviruses and is redundant when BrYV co-infects with PEMV 2. These observations provide a novel insight into the conserved function of P3a and its underlying mechanism in the systemic infection.