Functional Characterization of RNA Silencing Suppressor P0 from Pea Mild Chlorosis Virus.

Qian Sun,Yuanyuan Li,Chenggui Han,Jialin Yu,Cuiji Zhou,Ying Wang,Tianyu Zhao,Dawei Li,Tao Zhuo

doi:10.3390/ijms21197136

Qian Sun, Yuanyuan Li + Show 7 more

Open Access

https://doi.org/10.3390/ijms21197136

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Abstract

To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.

Highlights

RNA silencing is an antiviral immune mechanism in a variety of eukaryotes including fungi, plants, and invertebrates
To test whether P0PM is an RNA silencing suppressor, a green fluorescent protein (GFP) agroinfiltration assay was performed in Nicotiana benthamiana [51,52]
The empty vector pGD served as a negative control, and P0PL and P19TBSV were used as positive controls [43,54]

Summary

Introduction

RNA silencing is an antiviral immune mechanism in a variety of eukaryotes including fungi, plants, and invertebrates. Recent data have shown that VSRs use various strategies to interfere with different phases of the silencing pathway including initiation of RNA silencing, dicing of viral dsRNA, assembly of RISCs, and amplification by RDRs [16,17,18,19]. V2 from tomato yellow leaf curl virus (TYLCV) inhibits RDR6-mediated amplification by directly interacting with SGS3 [25] and P6 from cauliflower mosaic virus (CaMV), which effectively suppresses silencing by interacting with dsRNA-binding protein 4 (DRB4) [26]. P0 proteins, the VSRs of poleroviruses, have been shown to trigger the degradation of AGO1 via autophagy [31,32,33,34,35]. We investigated the ability of P0PM to suppress local and systemic RNA silencing, trigger AGO1 degradation, and interact with SKP1. We identified the critical amino acids responsible for P0PM VSR activity

P0PM Suppressed Local but Not Systemic RNA Silencing

Requirement of N- and C-Terminal Amino Acids of P0PM for Suppressor Activity

Plant Material and Growth Conditions

Agrobacterium-Mediated Transient Expression and GFP Imaging

Protein Extraction and Western Blots