Grapevine red blotch virus (GRBV) is a member of the Geminiviridae family, and infection of grapevines by this virus can lead to reduced fruit quality and yield. Viticultural practices and pest management strategies have been mainly ineffective at mitigating the impacts of GRBV, necessitating alternative control strategies. Integrated pest management strategies can be challenging to design, leaving growers with few alternatives. Current recommendations include using certified vines, monitoring and testing, and replacing infected grapevines.
In 2021, we developed a research program combining genomic and nanotechnology tools to propose an alternative strategy to combat GRBV. The concept is to spray a solution of specific double-stranded RNA molecules related to GRBV on the leaf surface, triggering RNA interference mechanisms (RNAi) in the grapevine against the virus. The use of particular nanomaterials can also help improve the uptake and the systemicity of this immune response. This approach aims to reduce the viral spread in infected plants and confer immunity to healthy plants. To be successful, one must first identify the region(s) in the genome of the GRBV virus that can be used to trigger immunity in grapevine, and second, characterize the appropriate nanomaterial(s) to ensure systemic silencing.
By artificially delivering GRBV to a grapevine and using Next Generation Sequencing, Christian Mandelli, a PhD student in the Deluc lab, identified nine genomic regions of the virus targeted by the RNA interference mechanism in grapevine (Mandelli and Deluc 2025). Afterward, he delivered the double-stranded RNA versions of these nine “hotspots” to trigger immunity in GRBV (+) grapevine, and he estimated the impact on the viral activity (Figure 1). The reduction of GRBV activity was sequence-specific because the delivery of dsRNA, by the root soaking method, to tissue-culture grapevine plantlets did not consistently result in reduced virus activity. However, about 40% of the tested hotspot-derived dsRNA showed viral silencing for at least one month after the infection.
Figure 1: Schematic illustration of identified hotspots (HS) across the GRBV genome of GRBV and their overlap with replication-(C) and structural(V)-associated genes (See Mandelli and Deluc, 2025). SIR: Short Intergenic Region, LIR: Long Intergenic Region.
One of the hotspots (HS9) was selected for an RNA spray experiment in greenhouse plants for its best ability to reduce GRBV activity. HS9 is located in a genomic region that encodes a protein involved in the replication of the virus. Following the in vitro production of the dsRNA, a solution of HS9-dsRNA was sprayed onto the lower surface of GRBV-positive grapevine leaves growing in greenhouse conditions. We implemented the procedure using a nanomaterial (Carbon Dots) that has been used previously to improve dsRNA delivery in the plant leaf surface (Schwartz et al. 2020). The choice of the lower surface of leaves to spray relies on several physical and biological features of plant leaves. The lower surface contains many more stomata, which serve as entry points for the sprayed dsRNA solution, allowing it to diffuse into the inner cells of the leaves. Usually, the lower surface cuticle is thinner and less waxy, which will facilitate a better diffusion of aqueous formulation. The lower surface is also relatively less exposed to evaporative stress and direct sunlight, allowing the sprayed droplets to remain longer on the surface.
Christian developed a cost-effective method to produce nanomaterial Carbon Dots, also called CDs, which have been demonstrated to facilitate the delivery of dsRNA in plants (Delgado-Martín et al. 2022, Schwartz et al. 2020). Following their synthesis, these CDs were tested for their capacity to bind dsRNA, and the proper dsRNA:CD ratio for internalization into grapevine intact cells (Figure 2). Then, Christian tested CDs in the context of silencing GRBV in grapevine-infected plants. To do so, he used the most promising hotspot region (HS9) identified from his Small RNA Seq data (Mandelli and Deluc 2025). GRBV-infected greenhouse plants were sprayed with a 20 nM solution of either naked HS9-dsRNA or complexed with CDs at a 1:200 dsRNA:CD mass ratio using a spray bottle. Three GRBV (+) plants per treatment group were used, with untreated plants sprayed with buffer used as control plants. To assess the systemic movement and silencing of HS9-dsRNA apart from the sprayed regions, unsprayed leaves of treated plants were collected at one node above and below the sprayed leaves (Figure 3A). After the spray treatment with naked and CD:dsRNA, total RNAs were extracted from both sprayed and unsprayed leaves one, two, and three months post-treatment. The same number of samples was collected for control plants.
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Christian performed qPCR analyses targeting the RepA GRBV gene, the supposed gene targeted by HS9-dsRNA (Figure 3B). The naked HS9-dsRNA (not complexed) showed a relatively strong reduction of RepA expression following the treatment after two months (90% of reduction), but this reduction was not maintained 3 months after the spray.
Figure 2: Characterization of Carbon Dots as a nanomaterial for delivering dsRNA in grapevine. A) Microwave-assisted synthesis of CDs (need some work from you). B) Physical characteristics of Carbon Dots. Left panel: blue luminescence of carbon dots under UV lights, Middle panel: Average size of the particle of Carbon dots, Right panel: Transmitted Electronic Microscopic image of isolated molecules of carbon dots (black dots), C) Intact grapevine cells treated with small interfering RNA tagged with Cy3 fluorophore with (upper panel) and without CDs (lower panel).
By contrast, spraying the HS9-dsRNA complexed with CDs significantly decreased the expression of the RepA gene compared to plants sprayed with buffer solution, and this reduction (95%) was maintained even three months post-spray. Interestingly, this reduction in RepA expression was observed between sprayed and unsprayed leaves (1 node above and below the sprayed leaves) from the treated plants in both conditions (naked versus CD-complexed dsRNA). This suggests effective systemic dsRNA transport following uptake from one node away to the treated leaves. Christian is currently attempting to replicate this result by analyzing leaves located two nodes away from the treated leaves.
Overall, the greenhouse experiment results are encouraging and need to be reproduced. The extent of silencing observed, in terms of magnitude and time, is significant compared to other studies that used either nano-clays or carbon-dots, which showed a more limited window of silencing efficacy over time (Mitter et al. 2017, Xu et al. 2023). Christian is currently repeating this experiment and conducting another experiment where he sprays several other dsRNA species corresponding to other potential hotspots. The integration of additional HS-derived dsRNA molecules, either from the virus or the grapevine, can be envisioned. We recently received an award from the California Department of Food and Agriculture to test the silencing of four grapevine genes, known as host factors, which were found to interact with GRBV proteins. This could be exploited by spraying several species of dsRNA to silence both the virus and the grapevine genes that help the virus spread.
Figure 3: Spray experiment on GRBV-infected grapevines using HS9-dsRNA: A) Location within treated plants of leaves collected to perform Real Time PCR, B) Upper panel: Gene expression of GRBV RepA gene (C1) in GRBV-infected plants following low-pressure spray with either naked HS9-dsRNA (upper panel) or CD-complexed HS9-dsRNA (lower panel) over three months post-spray. The statistical significance between control and treated plants over time has been tested with the Student’s t-test for a p-value < 0.05. The black error bars indicate the standard error of the mean (SEM), and the letters on top of each bar indicate the statistically different groups (p-value < 0.05) following Tukey’s HSD test. mps (month post-spray). Because we could not observe any differences in RepA expression between sprayed and unsprayed leaves, the treated samples combine sprayed and unsprayed leaves (one node above and below the sprayed leaves).
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Additional information: This research project was funded by the PD/GWSS Board from 2020 to 2021, the OWRI and the Erath Family Foundation from 2021 to 2023, and the Oregon Wine Board from 2023 to 2026. — By Dr. Laurent Deluc, Associate Professor, Department of Horticulture, Oregon State University, and Christian Mandelli, Ph.D. student, Department of Horticulture, Oregon State University.
Literature Cited:
Delgado-Martín J, Delgado-Olidén A, Velasco L. 2022. Carbon Dots Boost dsRNA Delivery in Plants and Increase Local and Systemic siRNA Production. International Journal of Molecular Sciences 23:5338.
Mandelli MC, Deluc PLG. 2025. Early Activation of RNAi Reveals Genomic Regions of Grapevine Red Blotch Virus Targeted for Silencing in Grapevine. https://doi.org/101094/MPMI-04-25-0038-R.
Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, Fletcher SJ, Carroll BJ, Lu GQ (Max), Xu ZP. 2017. Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants 3:1–10.
Schwartz SH, Hendrix B, Hoffer P, Sanders RA, Zheng W. 2020. Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants. Plant Physiology 184:647–657.
Xu X, Yu T, Zhang D, Song H, Huang K, Wang Y, Shen L, Li Y, Wang F, Zhang S, et al. 2023. Evaluation of the anti-viral efficacy of three different dsRNA nanoparticles against potato virus Y using various delivery methods. Ecotoxicology and Environmental Safety 255:114775.