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.

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.

Sweet cherry (Prunus avium L.) cultivation in China covers an estimated area of 25,600 hectares, representing more than one-third of the global total. Viral diseases present a serious challenge to cherry production worldwide; however, the phytosanitary status of sweet cherry in China has remained poorly understood. In this study, 191 sweet cherry samples were collected from major growing regions and screened using RT-PCR combined with DNA sequencing for the presence of 14 viruses previously reported in China. Results revealed that 80.1% of the tested samples were infected with at least one virus, with mixed infections detected in 51.3% of the samples. Prevalent viruses included cherry virus A (CVA, 53.4%), prunus necrotic ringspot virus (PNRSV, 35.1%), cherry green ring mottle virus (CGRMV, 32.5%), plum bark necrosis stem pitting-associated virus (PBNSPaV, 31.4%), and prune dwarf virus (PDV, 10.5%). Cherry necrotic rusty mottle virus (CNRMV) was found at a very low frequency (0.5%), and the remaining eight viruses were not detected in any sample. Based on these findings, we developed multiplex RT-PCR assays for simultaneous detection of CVA, PNRSV, CGRMV, PBNSPaV, and PDV. Several dual and triplex RT-PCR systems were successfully established, including combinations such as PBNSPaV/PNRSV, CVA/PDV, CVA/CGRMV, PBNSPaV/PDV/CGRMV, and PBNSPaV/PNRSV/PDV. This study identifies CVA, PNRSV, CGRMV, PBNSPaV, and PDV as the prevalent viruses in the investigated Chinese sweet cherry orchards. Accordingly, multiplex RT-PCR assays were developed for their simultaneous detection. Our work advances the understanding of sweet cherry viral diseases in China and provides a valuable complementary tool for the existing diagnostic toolkit.

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 also constructed. Infection assays indicated that Nicotiana benthamiana plants inoculated with GGVAQN display upward leaf curling and chlorotic mottling symptoms. Direct injection of Agrobacterium tumefaciens cells carrying pXT-GGVAQN into roots of in vitro-grown ‘Red Globe’ grape plantlets by syringe led to systemic infection 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.

Grapevine (Vitis vinifera) is severely impacted by viral diseases, with grapevine leafroll disease (GLRD) being the most economically damaging. Grapevine leafroll-associated virus 2 (GLRaV-2) is a prevalent virus associated with GLRD, yet the molecular mechanisms underlying GLRaV-2 infection remain poorly understood. Here, we demonstrate that the grapevine transcription factor (TF) VvWRKY51 positively autoregulates its transcription and directly represses salicylic acid (SA)-responsive defense genes VvPR1 (pathogenesis-related gene 1), VvPR2, and VvPR5. Through in vivo and in vitro assays, we demonstrate that GLRaV-2 RNA silencing suppressor p24 specifically interacts with VvWRKY51 to promote viral infection. p24 hijacks nuclear VvWRKY51 by translocating it to the cytoplasm, thereby enhancing its RNA silencing suppression activity via 2 mechanisms: (i) evading host degradation pathways (ubiquitin-proteasome system and autophagy), and (ii) strengthening its small interfering RNA-binding affinity. Concurrently, cytoplasmic p24 enters the nucleus by binding VvWRKY51, where it reinforces VvWRKY51-mediated transcriptional repression of VvPR1, VvPR2, and VvPR5. This occurs by enhancing VvWRKY51 binding to its own promoter and those of VvPR genes. Our findings reveal a novel viral strategy wherein a single viral suppressor of RNA silencing simultaneously suppresses RNA silencing and SA-mediated immunity by interacting with a host TF to establish a permissive environment for infection.