Abstract
Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24G1 may be a factor in pathogenesis. Furthermore, p24G1 function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122–139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.
Highlights
RNA silencing is considered to be the most effective antiviral mechanism in plants
We show that p24 of grapevine leafroll-associated virus 1 (p24G1) is a factor in pathogenesis eliciting a strong hypersensitive response (HR)-like response in Nicotiana species when it is expressed from a binary vector pGD–GFP with an empty vector (pGD) or potato virus X (PVX)-based vector
Our results revealed a correlation between the pathogenic activity of p24G1 and the upregulation of PR genes, supporting the notion that bioactive p24G1 induces a HR-like response
Summary
RNA silencing is considered to be the most effective antiviral mechanism in plants. Antiviral. The 2b protein encoded by cucumber mosaic virus (CMV) inhibits RNA silencing by binding ds siRNA [4] and interfering with RISC activity through interaction with AGO proteins [5,6]. Given the key role of RNA-silencing suppression for virus survival, many viral RSSs have been proposed to be pathogenicity or virulence factors in plants. Within the genus Ampelovirus, GLRaV-3 encodes one RSS, p19.7 [28]; pineapple mealybug wilt associated virus-2, which is closely related to GLRaV-1 and GLRaV-3, encodes four RSSs—p20, CP, p22 and CPd [29]. No such functional protein activity has been described for GLRaV-1. We evaluate the potential contribution of p24G1 homodimerization to its importin α1-mediated nuclear localization, binding of ds siRNA, silencing suppression function and activity in pathogenesis
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