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

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.

What we know about poleroviruses: Advances in understanding the functions of polerovirus proteins

“…in PLRV or I225 in PMCV) and W212 (tryptophan or W221 in PLRV or W2256 in PMCV; Han et al, 2010;Sun, Zhuo, et al, 2020;Zhuo et al, 2014).…”

“…However, those motifs are absent from P0, although this protein has a terminal carboxyl sequence (K/R)IYGEDGX3FWR (Figure 1) that could represent a type of novel substrate interaction domain (Pazhouhandeh et al, 2006). Analyses of mutants in this domain of the P0 protein of MABYV, PLRV, and pea mild chlorosis virus (PMCV) have revealed that the suppressor activity is mainly dependent on residues F211 (phenylalanine or F220 in PLRV or I225 in PMCV) and W212 (tryptophan or W221 in PLRV or W2256 in PMCV; Han et al, 2010; Sun, Zhuo, et al, 2020; Zhuo et al, 2014).…”

Comparative Analysis of Biological Characteristics among P0 Proteins from Different Brassica Yellows Virus Genotypes

“…To suppress the host anti-viral RNA silencing, many plant viruses have evolved viral suppressor of RNA silencing (VSR) proteins [ 2 , 3 , 4 , 5 ]. The P0 proteins have been identified as VSR in many poleroviruses, including Turnip yellows virus (TuYV), Cucurbit aphid-borne yellows virus (CABYV), Potato leafroll virus (PLRV), Beet mild yellowing virus (BMYV), Cotton leafroll dwarf virus (CLRDV), Sugarcane yellow leaf virus (ScYLV), Melon aphid-borne yellows virus (MABYV), Maize yellow mosaic virus (MaYMV), Cereal yellow dwarf virus (CYDV), Wheat yellow dwarf virus -GPV isolate (WYDV-GPV), Maize yellow dwarf virus-RMV 2 (MYDV-RMV2), and Pea mild chlorosis virus (PMCV) [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. The amino acid sequence of P0 proteins throughout the genus shows low identity and the RNA silencing suppressor (RSS) activity of the P0 proteins varies between different viruses.…”

“…Different isolates of the same virus (BMYV and PLRV) can exhibit various efficiencies in RSS activity [ 11 , 14 , 22 ]. P0 proteins of several polerovirus members can trigger ARGONAUTE1 degradation via the autophagy pathway to suppress RNA silencing [ 6 , 16 , 23 , 24 ]. Studies have shown that P0 proteins of TuYV, CABYV, CYDV-RPV, CYDV-RPS, and PMCV have a consensus F-box-like domain, through which P0 proteins interact with S-phase kinase-associated protein 1 (SKP1), a component of the SKP1–Cullin F box (SCF) family of the E3 ubiquitin ligases complex [ 10 , 18 ].…”

“…Studies have shown that P0 proteins of TuYV, CABYV, CYDV-RPV, CYDV-RPS, and PMCV have a consensus F-box-like domain, through which P0 proteins interact with S-phase kinase-associated protein 1 (SKP1), a component of the SKP1–Cullin F box (SCF) family of the E3 ubiquitin ligases complex [ 10 , 18 ]. Although PLRV and PMCV P0 proteins contain a conserved F-box-like domain and suppress RNA silencing, they fail to interact with SKP1 [ 6 , 11 ].…”