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Article

The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection

State Key Laboratory for Agrobiotechnology and Key Laboratory of Pest Monitoring and Green Management, MOA, China Agricultural University, Beijing 100193, China
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Author to whom correspondence should be addressed.
Viruses 2019, 11(2), 170; https://doi.org/10.3390/v11020170
Received: 22 January 2019 / Revised: 14 February 2019 / Accepted: 16 February 2019 / Published: 20 February 2019
(This article belongs to the Special Issue Plant Immunity to Virus Infections)
Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection. View Full-Text
Keywords: Potato leafroll virus; viral suppressor of RNA silencing; P0 mutation; F-box-like motif; G/W/G-like motif; C-terminal conserved region; virus infection Potato leafroll virus; viral suppressor of RNA silencing; P0 mutation; F-box-like motif; G/W/G-like motif; C-terminal conserved region; virus infection
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    Description: Figure S1: Infectivity analysis of full-length cDNA of pCa-PLRV and pCB-PLRV in N. benthamiana. (A) Symptoms on N. benthamiana leaves agro-infiltrated with pCB empty vector (negative control), pCa-PLRV and pCB-PLRV respectively, at 7 dpi. (B) Assessment of viral RNA accumulation in systemic leaves of inoculated N. benthamiana plants at 14 dpi by RT-PCR amplification with PLRV-specific primer pair. Figure S2: Impact analysis of additional heterologous expression of VSR (P0PL) on the infectivity of VSR defective mutants in F-box-like motif, G139/W140/G141-like motif and C-terminal conserved region derived from pCB-PLRV in N. benthamiana. (A) Symptoms on N. benthamiana leaves agro-infiltrated with pCB empty vector (negative control), wild-type pCB-PLRV (positive control) and mutants pCB-PL-L76F, pCB-PL-W87R, pCB-PL-G139RRR and pCB-PL-F220R respectively, in absence or presence of P0PL at 7 dpi. (B) Assessment of viral RNA and protein accumulation in N. benthamiana leaves inoculated with pCB-PLRV mutants in absence or presence of P0PL at 3 dpi by RT-PCR amplification and western blot analysis. (C) Assessment of viral RNA and protein accumulation in systemic leaves of N. benthamiana plants inoculated with pCB-PLRV mutants in absence or presence of P0PL at 14 dpi by RT-PCR amplification and western blot analysis. The upper panel shows the RT-PCR amplification with PLRV-specific primer pair. The middle panel shows the western blot analysis with the PLRV-MP specific antibody (anti-MPPLRV). Rubisco stained with Coomassie brilliant blue used as a loading control is shown in the lower panel. Table S1: Test results for RNA silencing suppression of P0PL and its mutants.
MDPI and ACS Style

Rashid, M.-O.; Zhang, X.-Y.; Wang, Y.; Li, D.-W.; Yu, J.-L.; Han, C.-G. The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection. Viruses 2019, 11, 170. https://doi.org/10.3390/v11020170

AMA Style

Rashid M-O, Zhang X-Y, Wang Y, Li D-W, Yu J-L, Han C-G. The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection. Viruses. 2019; 11(2):170. https://doi.org/10.3390/v11020170

Chicago/Turabian Style

Rashid, Mamun-Or, Xiao-Yan Zhang, Ying Wang, Da-Wei Li, Jia-Lin Yu, and Cheng-Gui Han. 2019. "The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection" Viruses 11, no. 2: 170. https://doi.org/10.3390/v11020170

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