Construction of an Infectious DNA Clone of Grapevine Geminivirus A Isolate GN and Its Biological Activity in Plants Analyzed Using an Efficient and Simple Inoculation Method.

The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVAQN) obtained from grapevine 'Queen Nina' plants with severe disease symptoms. The infectious clone of GGVAQN (pXT-GGVAQN) was constructed to investigate its pathogenicity. Nicotiana benthamiana plants inoculated with GGVAQN by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVAQN into grapevine plants was developed, by which Agrobacterium tumefaciens cells carrying pXT-GGVAQN were introduced into the roots of in vitro-grown 'Red Globe' grape plantlets with a syringe. By this method, all 'Red Globe' grape plants were systemically infected with GGVAQN, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.

GLRaV-2 protein p24 suppresses host defenses by interaction with a RAV transcription factor from grapevine.

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.