Identification of Strawberry vein banding virus encoded P6 as an RNA silencing suppressor

Abstract

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation.

Identification of the Potential Virulence Factors and RNA Silencing Suppressors of Mulberry Mosaic Dwarf-Associated Geminivirus

“…To explore which region(s) within MMDaV V2 is crucial for suppressing RNA silencing, we analyzed the MMDaV V2 sequence, and found that the RRLLRLIRRFSRVKDR motif between amino acid 61 and 76 defined a bipartite basic nuclear localization signal (NLS). Previous studies have indicated that the NLS of several VSRs is essential for the RNA silencing suppressor activity [ 31 , 37 , 38 , 39 ], prompting us to determine whether this motif is crucial for V2 to suppress PTGS. At a first step, we determined the subcellular localization of V2 via transient expression of eGFP-fused constructs into N. benthamiana leaves.…”

“…Despite the fact that deletion of the putative NLS from MMDaV V2 did not prevent translocation of the protein into the plant nucleus, mutagenesis experiment demonstrated that the predicted basic motif of MMDaV V2 is indispensable not only for subnuclear foci localization but also for silencing suppression. In previous studies, nuclear import was found mandatory for several VSRs, such as βC1 of tomato yellow leaf curl China betasatellite and tomato leaf curl China betasatellite, P37 of Pelargonium line pattern virus, and P6 of cauliflower mosaic virus (CaMV) and strawberry vein banding virus, to suppress RNA silencing [ 31 , 37 , 38 , 39 , 54 ]. Nuclear import of CaMV P6 is conducted via two importin-α-dependent NLSs, and it is required for CaMV infection and suppression of the nuclear RNA silencing factor dsRNA-binding protein 4 (DRB4) [ 54 ].…”

Rescue of Tomato spotted wilt tospovirus entirely from cDNA clones, establishment of the first reverse genetics system for a segmented (-)RNA plant virus

“…DIG-labeled specific probes for sense or antisense GFP, NSs, NSm, L-5’UTR was synthesized by DIG High Prime RNA labeling kit (Roche, Basel, Switzerland). The total RNAs were separated on 1 % formaldehyde agarose gels and transferred to Hybond-N+ membranes (GE Healthcare, UK) (8). The membrane blots were hybridized with a DIG-labeled specific probe and detected using a DIG-High Prime Detection Starter Kit II (Roche), following the manufacturer’s protocol.…”

Strawberry Vein Banding Virus P6 Protein Is a Translation Trans-Activator and Its Activity Can be Suppressed by FveIF3g

“…The CaMV ORF VI-encoded P6 protein was reported as a multifunctional protein [10]. For example, CaMV P6 can function as an RNA silencing suppressor, and is the main component of CaMV inclusion bodies [11,12]. It is also known to be a disease symptom and host range determinant [13].…”

“…Finally, the CaMV P6 protein was also shown to regulate the expression of multiple genes in its host plants [15]. In our previous studies, the SVBV P6 protein had also been identified as an RNA silencing suppressor, which could effectively enhance the expression of the exogenous GFP gene Nicotiana benthamiana [11].…”