Identification Of Amino Acid Sequence Motifs Essential For RNA Silencing Suppression In P0from An Inner Mongolia Isolate Of Potato Leafroll Virus

Potato leafroll virus (PLRV) is distributed worldwide and infects economically important potato crop, causing important loss in agriculture. PLRV which belongs to the Luteoviridae family, is the type member of the Polerovirus genus and the genome consists of single-stranded plus-sense RNA with six recognized ORFs (open reading frames). The PO proteins encoded by the5’-proximal ORF of poleroviruses are well known RNA silencing suppressors. Recently, many PO-related studies have been reported, but the mechanism of PO remains unclear. In our study, we used Agrobacterium infiltration-mediated RNA silencing assays to establish that the Inner Mongolian PLRV PO protein (POPL-IM) has a strong suppressor activity. Five amino-acid substitutions in POPL-IM with those corresponding to the PO protein of a Netherlandish isolate of PLRV (POPL-NL) did not affect the strong suppressor activity, implying that the distinction between the two PO proteins was independent of the differences between their amino acids. But the mutations to A among the different amino acids, S102A and I228A, affect its suppressor activity. Strikingly, poPL-IM has an unusual F-box-like motif that contains a Trp/Gly sequence (87W/88G) and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other PO proteins, and contains two POPL-IM LP motif signature residues, L59/P60and L76/P77, matched an F-box-like domain. Mutagenesis experiments demonstrated that the POPL-IM F-box-like motif encompasses amino acids76-LPRHLHYECLEWGLLCGTHP-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/G141region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of POPL-IM. We conducted the alignment of POPL from various isolates and found the motifs (76LPRHLHYECLEWGLLCGTHP95, G139WG140and F220) were very conserved and had no variation and the results of mutagenesis experiments also comfirmed these motifs are very important to POPL suppressor activities. As has been observed with other PO proteins, the suppression of POPL-IM is correlated with the reducion of the accumulation of host AGO1silencing complex protein. However, POPL-IM fails to bind Nicotiana benthamiana SKP1, which functions in a proteasome pathway that may be involved in AGO1degradation, either in yeast or in plants. These results suggest that P0PL-IM may suppress RNA silencing by using an alternative pathway to target AGO1for degradation.In our studies, we also identified another strong RNA silencing suppressor, the PO protein of Pea mild chlorisis virus (PMCV) and also found a F-box-like motif in PMCV PO. The LP motif signature residues, L62/P63, contributed to the suppressor activity. The PMCV PO is the longest known PO protein among polerovirues, which has271amino acids. Deletion of the first20N-terminal amino acid residues of PMCV PO destroyed suppression of RNA silencing, but the41C-terminal amino acid residue region of PMCV PO was not required for silencing suppressor activity.All of the results enrich the diversity of POs of poleroviruses as suppressors and help improve our understanding of the molecular mechanisms involved in poleroviruses infection.