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.

Tomato yellow leaf curl virus (TYLCV) is a prominent viral pathogen that adversely affects tomato plants. Effective strategies for mitigating the impact of TYLCV include isolating tomato plants from the whitefly, which is the vector of the virus, and utilizing transgenic lines that are resistant to the virus. In our preliminary investigations, we observed that the use of growth retardants increased the rate of TYLCV infection and intensified the damage to the tomato plants, suggesting a potential involvement of gibberellic acid (GA) in the conferring of resistance to TYLCV. In this study, we employed an infectious clone of TYLCV to inoculate tomato plants, which resulted in leaf curling and growth inhibition. Remarkably, this inoculation also led to the accumulation of GA₃ and several other phytohormones. Subsequent treatment with GA₃ effectively alleviated the TYLCV-induced leaf curling and growth inhibition, reduced TYLCV abundance in the leaves, enhanced the activity of antioxidant enzymes, and lowered the reactive oxygen species (ROS) levels in the leaves. Conversely, the treatment with PP333 exacerbated TYLCV-induced leaf curling and growth suppression, increased TYLCV abundance, decreased antioxidant enzyme activity, and elevated ROS levels in the leaves. The analysis of the gene expression profiles revealed that GA₃ up-regulated the genes associated with disease resistance, such as WRKYs, NACs, MYBs, Cyt P450s, and ERFs, while it down-regulated the DELLA protein, a key agent in GA signaling. In contrast, PP333 induced gene expression changes that were the opposite of those caused by the GA₃ treatment. These findings suggest that GA plays an essential role in the tomato’s defense response against TYLCV and acts as a positive regulator of ROS scavenging and the expression of resistance-related genes.

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.

Tomato yellow leaf curl virus (TYLCV) is a devastating pathogen that causes substantial yield losses, and this virus can infect both tomatoes (Solanum lycopersicum L.) and tobacco (Nicotiana benthamiana). In this study, a constructed infectious clone of TYLCV was used for the exploration of tomato and tobacco plants' response to virus infection. Infected plants exhibit typical symptoms of TYLCV, including leaf chlorosis, curling, and plant dwarfing. Reactive oxygen species accumulated, and severe cell necrosis appeared in the tomatoes and tobacco that were infected. After TYLCV infection, 6,775 and 900 genes’ expressions were up-regulated in tomatoes and tobacco, including MYB and MADS-box transcription factors, serine/threonine protein kinase, heat shock proteins, cytochrome P450s, E3 ubiquitin-protein ligase, RAV transcription factors. Several stress-responsive kinases involved in autophagy were significantly up-regulated in tobacco but not in tomato. Moreover, silencing the RAV transcription factor, which is associated with the salicylic acid induced antiviral signaling, led to decreased virus abundance in tomato leaves. The results are helpful for an in-depth understanding of plants’ resistance to TYLCV infection.

Introduction: The BASIC PENTACYSTEINE (BPC) family comprises plant-specific transcription factors that regulate diverse developmental programs and stress responses. Pear ( Pyrus bretschneideri ), an economically significant fruit crop, often experiences marked declines in fruit yield and quality under drought stress. Although BPC genes have been identified in several plant species, a comprehensive characterization of this family in pear is lacking. Methods: In this study, we systematically characterized PbBPC genes in the pear genome using various bioinformatic approaches. We examined their expression profiles across diverse tissues and under dehydration conditions and further validated the role of PbBPC5 in drought tolerance using virus-induced gene silencing (VIGS). Results: This study identified seven PbBPC genes in the pear genome, which were subsequently classified into three distinct groups through phylogenetic analysis. Comprehensive bioinformatics analyses were performed, examining their phylogenetic relationships, gene structures, conserved motifs, protein domains, chromosomal locations, and gene duplication events. Promoter analyses showed that all PbBPC genes contained various cis -acting elements associated with growth and development, stress response, and phytohormone signaling. Quantitative real-time PCR (qRT-PCR) showed that most PbBPC transcripts were upregulated by dehydration, with PbBPC5 exhibiting the strongest upregulation. Furthermore, subcellular localization experiments indicated that PbBPC5 was localized to the nucleus. Silencing PbBPC5 reduced drought tolerance, as indicated by more severe wilting under water deficit, lower relative water content, higher electrolyte leakage, and elevated malondialdehyde levels. PbBPC5 silencing also weakened antioxidant defenses during drought by reducing antioxidant enzyme activities. These results suggest that PbBPC5 functions on drought tolerance regulation in pear mainly by influencing reactive oxygen species scavenging. Discussion: This study provides a genome-wide characterization of the PbBPC family and reveals PbBPC5 as a key regulator of the drought response, offering a foundation for improving pear drought tolerance through genetic approaches.

Tomato yellow leaf curl virus (TYLCV) is a widely distributed pathogen responsible for tomato yellow leaf curl disease. However, the molecular mechanism underlying TYLCV infection and the plant antivirus pathway remains unclear. This study reports that tomato ( Solanum lycopersicum ) RELATED TO ABSCISIC ACID INSENSITIVE3/VIVIPAROUS2 (SlRAV2), a transcription factor of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) superfamily containing an AP2/B3 DNA‐binding domain, suppresses systemic TYLCV accumulation by interacting with the V2 protein, an RNA silencing suppressor (RSS) encoded by TYLCV. Transient overexpression and silencing of SlRAV2 altered TYLCV levels in systemic leaves, prompting us to investigate the underlying mechanism. Molecular analyses revealed that SlRAV2 directly binds to the promoter of the pathogenesis‐related protein 1 ( SlPR1 ) gene, thereby activating its expression and contributing to basal defence responses. V2 affects the nuclear import of SlRAV2 through protein–protein interaction and recruits SlRAV2 to bind more 21‐nt double‐stranded siRNA, thereby enhancing its own RSS activity and transiently increasing local TYLCV accumulation. Moreover, nuclear‐localised V2 increases SlRAV2 binding affinity for the SlPR1 promoter, resulting in elevated SlPR1 transcription and concomitant accumulation of reactive oxygen species (ROS). Our findings reveal a novel antiviral mechanism whereby SlRAV2 interacts with V2 to enhance the local accumulation of TYLCV transiently, causing localised cell necrosis and preventing viral spread from infiltrated leaves to systemic leaves, thereby suppressing the systemic infection of TYLCV.