Grapevine leafroll-associated virus 2 (GLRaV-2) is a prevalent virus associated with grapevine leafroll disease, but the molecular mechanism underlying GLRaV-2 infection is largely unclear. Here, we report that 24-kDa protein (p24), an RNA-silencing suppressor (RSS) encoded by GLRaV-2, promotes GLRaV-2 accumulation via interaction with the B3 DNA-binding domain of grapevine (Vitis vinifera) RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 (VvRAV1), a transcription factor belonging to the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily. Salicylic acid-inducible VvRAV1 positively regulates the grapevine pathogenesis-related protein 1 (VvPR1) gene by directly binding its promoter, indicating that VvRAV1 may function in the regulation of host basal defense responses. p24 hijacks VvRAV1 to the cytoplasm and employs the protein to sequester 21-nt double-stranded siRNA together, thereby enhancing its own RSS activity. Moreover, p24 enters the nucleus via interaction with VvRAV1 and weakens the latter’s binding affinity to the VvPR1 promoter, leading to decreased expression of VvPR1. Our results provide a mechanism by which a viral RSS interferes with both the antiviral RNA silencing and the AP2/ERF-mediated defense responses via the targeting of one specific host factor.

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 (p24^G1) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24^G1 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24^G1 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24^G1 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24^G1 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24^G1 may be a factor in pathogenesis. Furthermore, p24^G1 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 p24^G1. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.