Amino Acid Sequence Motifs Essential for P0-Mediated Suppression of RNA Silencing in an Isolate of Potato leafroll virus from Inner Mongolia

Abstract

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.

Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein

“…By mutagenizing the LP residues of either of the two motifs, the authors demonstrated that only the second motif was essential to block RNA silencing. Despite this, and unlike our observations, Zhuo et al (2014) were unable to show any interaction between P0 PL-IM and NbSKP1 (N. benthamiana ortholog of ASK1), suggesting that ubiquitination by a P0 PL-IM -containing SCF-type E3 ligase was not required for AGO1 degradation. A second imperfect F-boxlike motif was found in both P0 RPV and P0 RPS at the same position as the downstream F-box motif of P0 PL-IM (Fig.…”

“…Intriguingly, although P0 PL-IM shares poor amino acid conservation with P0 RPV and P0 RPS , the sequence alignment highlights the first WG motif in common (Fig. 6) that was determined to play an essential role in suppression of RNA silencing of the P0 PL-IM (Zhuo et al, 2014). This could point to another functional domain in CYDV P0s that may participate in a suppressor-related function, for instance in AGO1 binding.…”

“…For instance P0 encoded by some isolates of beet mild yellowing virus (BMYV) were reported to lack silencing suppression activity while other isolates of BMYV were fully competent in RNA silencing inhibition (Klein et al, 2014;Kozlowska-Makulska et al, 2010). Similarly Pfeffer et al (2002) reported a weak SSP activity for P0 encoded by a Dutch isolate of potato leafroll virus (PLRV), while Fusaro et al (2012) and Zhuo et al (2014) showed that P0 of an Australian and an Inner-Mongolian isolate respectively were strong inhibitors of RNA silencing. Finally, other polerovirus-encoded P0 proteins like those of beet chlorosis virus (four isolates tested) totally lacked such SSP function in an Fig.…”

“…This difference might explain the weaker interaction observed between P0 RPS and ASK2. Interestingly P0 of PLRV-IM (P0 PL-IM ) was reported to display a second F-box-like motif 15 amino acids downstream of the first one that is closer to the F-box consensus motif (Zhuo et al, 2014). By mutagenizing the LP residues of either of the two motifs, the authors demonstrated that only the second motif was essential to block RNA silencing.…”

“…LP residues in the F-box motif of P0 Tu are required for both SSP activity and ASK interaction (Baumberger et al, 2007). Conversely although the second F-box motif of P0 PL-IM was shown to be essential to block RNA silencing, P0 PL-IM failed to interact with NbSKP1 (Zhuo et al, 2014). This suggests the existence of overlapping functional domains in P0 proteins, one essential for silencing suppression and the other involved in ASK interactions.…”