Inhibition of Hop Stunt Viroid by Exogenous Double-Stranded RNA in Micropropagated Grapevine Plantlets

Chae-Min Kang; Rae-Dong Jeong

doi:10.5423/PPJ.OA.05.2025.0071

Plant Pathol J > Volume 41(4); 2025 > Article

Kang and Jeong: Inhibition of Hop Stunt Viroid by Exogenous Double-Stranded RNA in Micropropagated Grapevine Plantlets

Research Article

The Plant Pathology Journal 2025;41(4):507-517.

Published online: August 1, 2025

DOI: https://doi.org/10.5423/PPJ.OA.05.2025.0071

Inhibition of Hop Stunt Viroid by Exogenous Double-Stranded RNA in Micropropagated Grapevine Plantlets

Chae-Min Kang, Rae-Dong Jeong^*

Department of Applied Biology, Chonnam National University, Gwangju 61185, Korea

^*Corresponding author. Phone) +82-62-530-2075, FAX) +82-62-530-2069, E-mail) jraed2@jnu.ac.kr

Handling Editor : Eui-Joon Kil

(Received on May 26, 2025; Revised on June 26, 2025; Accepted on July 9, 2025)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Hop stunt viroid (HSVd) infection reduces grapevine (Vitis vinifera) yield and quality, leading to significant economic losses. Conventional methods for producing virus-free plantlets often fail to completely eliminate viroids, necessitating alternative strategies. In this study, RNA interference (RNAi) was induced by applying HSVd-specific double-stranded RNA (dsRNA) to infected grapevine plantlets. Exogenous dsRNA treatment significantly reduced HSVd levels, as confirmed by reverse transcription polymerase chain reaction and digital PCR. Fluorescently labeled (Cy3) dsRNA uptake was detected in plant tissues, while small RNA sequencing revealed an accumulation of HSVd-derived small interfering RNA, indicating RNAi activation. Notably, the inhibitory effect persisted through three successive generations without additional treatment, and similar suppression was observed in HSVd-infected cucumber plants. These findings highlight the efficacy and durability of exogenous dsRNA applications as a sustainable and non-transgenic approach for viroid control in grapevine cultivation.

Keywords: double-stranded RNA, hop stunt viroid, viroid-free grapevine plantlet, Vitis vinifera

Grapevine (Vitis vinifera) is among the most economically important fruit crops worldwide, extensively cultivated for wine production, table grapes, and raisins (Alston and Sambucci, 2019). However, viral and viroid infections, including grapevine fleck virus (GFkV), grapevine leafroll-associated virus (GLRaV), hop stunt viroid (HSVd), grapevine asteroid viroid (GAV), and grapevine yellow speckle viroid (GYSVd), present considerable threats to grape production, leading to reduced yields, inferior fruit quality, and substantial economic losses (Belkina et al., 2023; Di Serio et al., 2017). HSVd infection is of particular concern among these pathogens, as it induces severe symptoms, including stunting, chlorosis, loss of vigor, and a reduction in flower cone metabolite quality (e.g., alpha acids), ultimately impacting productivity and vineyard longevity. Owing to its extensive prevalence, HSVd continues to pose a significant challenge for viticulture (Kawaguchi-Ito et al., 2009; Sasaki and Shikata, 1977).

HSVd is a circular, single-stranded RNA viral pathogen that infects a wide range of plant species, including grapevine. Its infection can lead to substantial economic losses by reducing fruit yield and quality. HSVd spreads via multiple transmission routes, including mechanical inoculation, grafting, and seed propagation, complicating control efforts (Singhal et al., 2021). Unlike conventional viruses, viroids do not encode proteins but instead rely entirely on the host cell’s machinery for replication and systemic movement. Their distinctive structural characteristics and replication mechanism render effective management particularly difficult (Flores et al., 2008; Sano and Ishiguro, 1998). Conventional control methods, including stringent quarantine measures and the use of certified virus-free plant material, help limit HSVd spread but do not offer a direct means of eliminating the pathogen once infection occurs (Flores et al., 2005; Kovalskaya and Hammond, 2014). Consequently, ensuring the production of healthy propagation materials is essential in grapevine cultivation, underscoring the pressing necessity for innovative strategies to combat HSVd.

Traditionally, meristem culture, thermotherapy, and shoot tip grafting have been utilized in producing virus- and viroid-free planting material. While these methods effectively reduce pathogen presence, they are labor-intensive, time-consuming, and may not completely eliminate viroids such as HSVd (El-Dougdoug et al., 2010; Hu et al., 2021; Nuzzo et al., 2022). Additionally, the risk of reinfection via vectors or environmental sources continues to pose a considerable challenge. These limitations underscore the pressing need for innovative and more efficient strategies to manage HSVd infection in grapevines.

RNA interference (RNAi) has emerged as a promising approach for controlling plant RNA pathogens. Double-stranded RNA (dsRNA) is a crucial component of such approaches, as it triggers the degradation of complementary RNA sequences through the host’s gene-silencing mechanism (Laisney et al., 2020). RNAi is a sequence-specific gene silencing mechanism that relies on dsRNA processing into small interfering RNA (siRNA) via the activity of host Dicer-like enzymes. These siRNAs bind to the RNA-induced silencing complexes (RISC) to target and degrade complementary target RNAs, thereby inhibiting viral or viroid replication (Bocos-Asenjo et al., 2025; Chen et al., 2025). Recent studies have demonstrated that the exogenous application of dsRNA targeting viral or viroid RNA can effectively suppress pathogen replication in a wide range of plant species. Furthermore, dsRNA-based approaches provide an environmentally friendly alternative to chemical treatments and genetic modifications, rendering them an attractive option in sustainable agricultural practices (Laisney et al., 2020; Schwartz et al., 2020; Xu et al., 2023). HSVd is characterized by a highly stable secondary structure that affects its replication and interactions with the host’s RNA-silencing machinery (Marquez-Molins et al., 2021). Therefore, optimizing target region selection for dsRNA design is crucial to maximize silencing efficiency and efficacy.

In this study, an HSVd-specific dsRNA was designed and applied to infected grapevine plantlets to assess its effectiveness in suppressing viroid replication. Our results provide insights into the implementation of RNAi-based approaches for viroid disease management and highlight the potential of dsRNAs as an innovative technology for safeguarding grapevines against HSVd infection. Additionally, this study demonstrates the broader applicability of dsRNA-based strategies for controlling persistent viroid infections in agricultural crops, paving the way for more effective plant disease management.

Materials and Methods

Preparation of plant material and RNA extraction

Single infection by HSVd in micropropagated grapevine (Vitis vinifera cv. ‘188.08’) plantlets was confirmed using Oxford Nanopore sequencing, provided by the National Institute of Horticulture Science, Republic of Korea (Kang and Jeong, 2025). The infected plantlets were cultured in vitro on a medium supplemented with activated charcoal. The medium, consisting of 2.2 g/L Linsmaier & Skoog medium, 2 g/L activated charcoal, 10 g/L sucrose, and 9 g/L plant agar, was adjusted to pH 5.8 and subsequently autoclaved at 121°C for 15 min. Cultured grapevines were maintained in a growth chamber under a 16/8 h light/dark photoperiod with a light intensity of 2000 lx. Total RNA was extracted from grapevine plantlet leaves using the Super Plant RNA Extraction kit (In VirusTech Co., Gwangju, Korea) following the manufacturer’s instructions. The RNA quality and quantity of the extracted samples were assessed using a spectrophotometer (BioDrop, Biochrom Ltd., Cambridge, UK).

Detection of HSVd using reverse transcription polymerase chain reaction

HSVd presence in grapevine plantlets was confirmed by reverse transcription polymerase chain reaction (RT-PCR) using the SuPrimeScript RT-PCR premix (Genet Bio, Daejeon, Korea) following the manufacturer’s instructions. The RT-PCR reaction mixture (20 μL total volume) was prepared by combining 10 μL of SuPrimerScript RT Premix (Genet Bio), 2 μL of RNA template, 2 μL of each 10 μM forward and reverse primers (Kang and Jeong, 2025), and 6 μL of DEPC-treated water. Amplification was carried out under the following thermal cycling conditions: reverse transcription at 50°C for 30 min, initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 60 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. The RT-PCR products were then separated via electrophoresis on 1.5% agarose gels stained with RedSafe nucleic acid staining solution (iNtRON Biotechnology Inc., Seongnam, Korea) and visualized under UV light.

HSVd target sequences for dsRNA production

The complete genomic sequence of HSVd was amplified from total RNA using RT-PCR. The amplified HSVd sequence was cloned into the XbaI and HindIII sites of the pL4440 vector, containing two convergent T7 polymerase promoters oriented in opposite directions separated by multiple cloning sites. The recombinant plasmids were transformed into Escherichia coli HT115 (DE3) using the standard CaCl₂ transformation protocol. E. coli HT115 (DE3) is an RNase III-deficient E. coli strain engineered to express T7 RNA polymerase under the control of an IPTG-inducible promoter. This strain contains the T7 RNA polymerase gene on its chromosome under the lac promoter and lacks RNase III activity, making it suitable for dsRNA production.

In vivo production of dsRNA in E. coli HT115

A single colony of E. coli HT115 (DE3) harboring the recombinant pL4440 plasmid was cultured in LB medium supplemented with 100 μg/mL ampicillin and 12.5 μg/mL tetracycline at 37°C for 16 h. The overnight culture was then subcultured in 200 mL of YT broth under identical conditions until the optical density at 595 nm (OD₅₉₅) reached 0.4. The expression of T7 polymerase was induced by adding IPTG, followed by incubation of the culture with shaking at 37°C for an additional 2 h. For dsRNA extraction, a phenol-chloroform purification step was performed prior to ethanol precipitation. E. coli cells were harvested by centrifugation at 4,000 ×g in a Falcon tube, resuspended in 1 mL of Trizol using a vortex, and mixed with 0.2 mL of chloroform. The samples were then centrifuged at 12,000 ×g for 15 min at 4°C to separate the aqueous and organic phases. The supernatant was transferred to a new tube, and the nucleic acids were precipitated using cold isopropanol. The resulting nucleic acid pellet was resuspended in DEPC-treated water and treated with RNase T1 at 37°C for 15 min to remove single-stranded RNA, yielding dsRNA to be used for HSVd inhibition.

Application of HSVd-dsRNAs in grapevine plantlets

The extracted HSVd-dsRNA was adjusted to 500 μg/mL, utilizing 1 mL for each spray application. All treatments were performed under sterile conditions on a clean bench. In vitro grapevine plantlets were sprayed at 7-day intervals, and samples were collected 28 days post-treatment (dpt). HSVd accumulation was assessed before and after treatment using RT-PCR and RT-digital PCR (RT-dPCR) with HSVd-specific primers and a probe. Mock-treated plants (DEPC-treated water) were used as controls.

Fluorescent labeling and observation of dsRNA uptake

Cy3-labeled dsRNA was synthesized using the in vitro Transcription T7 Kit (D6140, TaKaRa, Tokyo, Japan) following the manufacturer’s instructions. The labeled dsRNA was then purified using the GeneJET Gel Extraction Kit (K0692, Thermo Scientific, Waltham, MA, USA), and its final concentration was adjusted to 0.5 mg/mL. The fluorescently labeled dsRNAs were applied to grapevine plantlets via spraying. After 24 h of spray treatment, leaf samples were collected and examined via confocal microscopy to evaluate dsRNA uptake.

Digital PCR analysis of the inhibitory effect of the applied dsRNAs on HSVd in micropropagated grapevine plantlets

The inhibition efficiency of dsRNA treatment against HSVd in in vitro grapevine plantlets was evaluated by quantifying HSVd levels using RT-dPCR. Total RNA quality was assessed with a BioDrop spectrometer (Biochrom, Ltd., Cambridge, UK), and nanoplate-based RT-dPCR was performed using HSVd-specific primers and probes (Supplementary Table 1). RT-dPCR was conducted using the QIAcuity One 2-plex digital PCR system (Qiagen, Hilden, Germany). The reaction mixture was prepared to a final volume of 40 μL, containing 10 μL of 4× QIAcuity Probe Master mix (Qiagen), 900 nM of the forward and reverse primers, 250 nM of the probe, and RNA template, following the manufacturer’s instructions. The prepared reaction mixture was then loaded into QIAcuity 26k 24-well Nanoplates (Qiagen) and sealed using the Qiagen standard priming profile. The RT-dPCR protocol included an enzyme activation step at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. Fluorescent images were captured with an exposure time of 500 ms (FAM channel), and segmentation data were analyzed using QIAcuity Suite Software V2.0.20.

HSVd-specific siRNA analysis by high-throughput sequencing

Small RNA sequencing was performed on HSVd-infected and dsRNA-treated grapevine leaves. Total RNA quality was assessed with a BioDrop spectrometer (Biochrom, Ltd.), and libraries were constructed using the SMARTer Small RNA Library Preparation Kit (Takara Bio USA, Inc., San Jose, CA, USA) following the manufacturer’s instructions. Total RNA was polyadenylated for oligo(dT) priming and was followed by first-strand cDNA synthesis performed using PrimeScript Reverse Transcriptase and the 3′ smRNA dT Primer. Non-templated nucleotides added to the 3′ end enabled SMART smRNA Oligo binding, facilitating template switching and extension. Full-length Illumina adapters carrying sample-specific indexes were added via PCR. The amplified small RNA libraries were purified using the NucleoSpin Gel and a PCR clean-up kit, followed by further enrichment using the AMPure XP Reagent (Beckman Coulter, Brea, CA, USA). DNA fragments were eluted in 22 μL of 10 mM Tris Buffer (pH 8.5), and the concentration was measured using the Qubit HS DNA kit (Life Technologies, Carlsbad, CA, USA). Sequencing was conducted on the Illumina platform by Macrogen (Seoul, Korea).

Small RNAseq data analysis

Generated reads ranging from 18 to 32 nt were selected for quality control, and the length distribution of siRNAs was calculated. Illumina adapters were removed and trimmed using Geneious Prime (version 2025.1.2). Initial quality filtering was conducted on Ubuntu (version 20.04) to remove poly-A sequences and reads shorter than 20 bp. High-quality siRNA reads were then aligned to the full-length HSVd genome using Geneious Prime. The analysis focused on identifying 21-24 nt siRNAs, which are the major constituents of the RNAi pathway. Small RNA expression was normalized per unit length of the HSVd genome based on alignment results.

Evaluation of the inhibitory effect of dsRNA on HSVd infection in cucumber plants

Cucumber seedlings transplanted into the soil were grown in a controlled environment at 25°C, 65% relative humidity, and a 16/8 h light/dark cycle. At the 3rd-4th true leaf stage, plants were inoculated with HSVd by gently rubbing 20 μL of inoculum—prepared by grinding 0.25 g of HSVd-infected grapevine plantlets in 1 mL of phosphate buffer—onto carborundum-doused leaves. One hour later, 1 mL of HSVd-specific dsRNA (300 μg/mL) was sprayed onto the leaves. Plants inoculated with HSVd without receiving dsRNA treatment were used as controls, while mock-inoculated plants (phosphate buffer only) served as negative controls.

Results

Production of dsRNAs targeting HSVd

To obtain dsRNAs specifically targeting HSVd in HT115 cells, RNA was extracted using the TriZol method after 6 h of IPTG induction. The designated dsRNAs were obtained from HT115 cells carrying the L4440-dsRNA plasmid. After IPTG induction, total RNA was obtained, and RNase T1 treatment was used to remove single-stranded RNA (Supplementary Fig. 1).

Time-dependent inhibition of HSVd by dsRNA treatment

Grapevine plantlets were treated with 1 mL of HSVd-specific dsRNA (500 μg/mL), and HSVd accumulation was assessed at 2, 4, 6, and 8 dpt using RT-PCR with HSVd-specific primers. A progressive reduction in HSVd levels was observed starting at 4 dpt, with no discernible HSVd bands detected by gel electrophoresis at 6 and 8 dpt. Conversely, control plantlets treated with DEPC water showed no reduction in HSVd accumulation (Fig. 1A). RT-dPCR was then conducted utilizing identical RNA templates to quantify HSVd levels with greater precision. In accordance with RT-PCR results, HSVd concentrations gradually decreased, falling below 100 copies by 6 and 8 dpt (Fig. 1B, Supplementary Table 2). These results indicate a significant reduction in HSVd accumulation starting from day 6 following a single dsRNA treatment. This experiment was independently repeated three times, and all replicates yielded similar results.

Suppression of HSVd in in vitro micropropagated grapevine plantlets by dsRNA treatment

To evaluate the inhibitory effect of exogenous dsRNA application on HSVd in in vitro micropropagated grapevine plantlets, 50 HSVd-infected plants were treated with dsRNA over a 28-day period. Each culture container, containing three plants, was sprayed weekly with 1 mL of dsRNA solution at a concentration of 500 μg/mL (Fig. 2A). HSVd amplification was reduced in all treated plants compared to 0 dpt based on RT-PCR analysis, whereas HSVd levels remained unchanged in DEPC-treated controls (Fig. 2B). A consistent decrease in HSVd concentration was further confirmed by RT-dPCR in all treated samples (Fig. 2C). Notably, 23 plants exhibited a reduction in HSVd levels by more than 70%, and HSVd became undetectable in 30% of the total population after treatment (Supplementary Table 3). No signs of growth inhibition, proliferation-related toxicity, or contamination were observed in any dsRNA-treated plants throughout the 28-day treatment period (Fig. 2A).

Spatial distribution and uptake of Cy3-labeled dsRNAs in grapevine plantlets

The uptake of dsRNAs in grapevine plantlets was assessed using Cy3-labeled dsRNA (Fig. 3A). A 1 mL solution of Cy3-dsRNAs (500 μg/mL) was applied to the plantlets by spraying. No fluorescence signal was detected in DEPC-treated control plants, confirming the absence of dsRNA uptake. In contrast, strong red fluorescence was observed in dsRNA-treated plantlets, particularly in leaf cells and along the veins, indicating successful uptake and potential vascular transport (Fig. 3B). The fluorescence signal and the localization of dsRNAs were further confirmed by the merged images and brightfield microscopy, supporting its cellular entry and movement within the plant (Fig. 3C). These results demonstrate that exogenously applied dsRNA can be absorbed and systemically distributed in grapevine plantlets.

Validation of the dsRNAs effects on HSVd suppression in cucumber plants

To assess the efficacy of HSVd-targeting dsRNA in other plant species, cucumber plants, a natural host of HSVd, were tested. Plants were inoculated with HSVd and then sprayed with 1 mL of dsRNA (500 μg/mL) 1 h post-inoculation. Disease symptoms were evaluated at 28 days post inoculation (dpi). HSVd-inoculated plants displayed dwarfism and stunted growth when compared to non-inoculated controls, whereas dsRNA-treated plants exhibited normal development and remained symptom-free (Fig. 4A). The experiments were independently replicated twice, each in triplicate, yielding consistent results. HSVd was detected exclusively in plants that were not inoculated with dsRNA, as shown by the RT-PCR analysis. Conversely, no amplification was observed in plants that were treated with dsRNA (Fig. 4B). These findings demonstrate that exogenous dsRNAs can effectively inhibit HSVd infection in cucumber plants.

Analysis of the siRNA molecules from the HSVd genome

To investigate the mechanism of HSVd suppression by exogenous dsRNA application, 21-24 nt siRNA profiling was performed in grapevine plantlets treated with HSVd-specific dsRNAs. siRNA reads were mapped to the HSVd genome, and their distribution and abundance were compared between untreated and dsRNA-treated samples. In untreated plants, siRNAs were sparsely distributed, with the sense strand exhibiting a higher accumulation, while moderate accumulation was observed for the antisense strand. Overall siRNA abundance was low. Conversely, dsRNA-treated plants showed a marked increase in siRNA coverage across the entire HSVd genome (Fig. 5A), with high siRNA levels detected in both sense and antisense orientations. These results indicate that exogenous dsRNA effectively activated the RNAi pathway, promoting the production of HSVd-specific siRNAs.

Furthermore, analysis of HSVd-derived siRNA size distribution revealed that the dsRNA-treated grapevine plants accumulated a significantly higher number of 21-, 22-, 23-, and 24 nt siRNAs compared to untreated controls. Among these, 22 nt siRNAs were the most abundant, followed by 21 nt siRNAs (Fig. 5B). These results confirm that exogenous application of HSVd-specific dsRNA effectively induces the production of viroid-targeting siRNA, promotes post-transcriptional gene silencing, and induces the expression of HSVd replication in grapevine plantlets.

Efficacy of HSVd suppression in in vitro regenerated grapevine plantlets

Shoot tips from dsRNA-treated grapevine plantlets were excised and cultured under standard growth conditions (Fig. 6A). To assess the persistence of HSVd suppression, HSVd levels in regenerated plants were quantified via RT-PCR and RT-dPCR over three successive generations. All third-generation grapevine plantlets exhibited HSVd concentrations that were either comparable to or lower than those observed at 28 dpt (Fig. 6B and C, Supplementary Table 4). These results demonstrate that the antiviral efficacy of dsRNA against HSVd can be sustained in regenerated grapevine plantlets without the need for additional dsRNA applications.

Discussion

dsRNAs have emerged as a promising tool for controlling plant viruses and viroids by harnessing the RNAi pathway—a conserved, sequence-specific gene silencing mechanism that degrades target RNA via siRNA. This approach offers a non-transgenic and environmentally friendly alternative for disease management, especially through exogenous dsRNA application or spray-induced gene silencing (SIGS) (Guo et al., 2019; Bradamante et al., 2021).

In this study, we demonstrated that exogenous application of HSVd-specific dsRNAs significantly inhibited HSVd replication in micropropagated grapevine plantlets. Small RNA analysis confirmed the activation of the RNAi pathway, corroborating the mechanism by which dsRNA is processed into siRNAs that guide RISC to degrade HSVd RNA (Bocos-Asenjo et al., 2025; Chen et al., 2025). The detection and profiling of HSVd-derived siRNAs indicated that the applied dsRNAs were efficiently processed and triggered the RNAi response (Donaire et al., 2009). Furthermore, siRNA distribution analysis across the HSVd genome revealed potential hotspots for cleavage, offering insights into the efficacy of different target regions.

A previous study demonstrated that HSVd infection in grapevine leads to the generation of abundant HSVd-derived siRNAs, particularly 21-, 22-, and 24 nt species, originating from both the (+) and (-) strands of the HSVd genome (Navarro et al., 2009). These findings suggest the involvement of host Dicer-like proteins (DCLs) in processing viroid RNAs during infection. In accordance with their observations, small RNA sequencing conducted in our study demonstrated that the exogenously applied HSVd-dsRNAs resulted in the accumulation of siRNAs of similar classes, suggesting that the host RNAi machinery was effectively activated. Furthermore, the siRNA distribution across the HSVd genome in our study revealed an overrepresentation of specific structural domains or replication intermediates of the viroid. The prevalence of 21- and 22 nt siRNAs in our samples also suggests the active engagement of DCL4 and DCL2, key components in antiviral defense. Collectively, these results support a model in which externally applied dsRNA mimics natural viral intermediates, triggering robust RNA silencing, leading to durable HSVd suppression.

Recent studies have emphasized the significance of RNAi in plant defense against viral invasion and transmission. SIGS, a non-transgenic RNAi approach, has attracted interest due to its safety, environmental friendliness, broad host range, and high specificity (Bocos-Asenjo et al., 2025; Parise et al., 2024; Vetukuri et al., 2021). This method enables the direct application of dsRNAs without requiring genetic transformation. Building on these advantages, we assessed dsRNA treatment as a viable and scalable approach for controlling HSVd in grapevine plantlets. Uptake of dsRNA was validated by Cy3 labeling and confocal microscopy, confirming its absorption within plant tissue. However, further studies are needed to elucidate the dynamics of dsRNA transport and persistence in grapevine.

RT-PCR and RT-dPCR were employed to evaluate the effectiveness of dsRNA treatment. While RT-PCR and RT-qPCR provide relative quantification of viroid RNA, dPCR enables absolute quantification with higher precision (Hindson et al., 2011; Luigi and Faggioli, 2011; White et al., 2009). A significant reduction in HSVd RNA levels was observed following dsRNA application, supporting its effectiveness as an antiviral strategy. These findings highlight the value of dPCR as a sensitive and reliable tool for assessing RNAi-mediated viroid control. Its high sensitivity also suggests potential utility in the production of viroid-free plants, reinforcing the applicability of dsRNA in sustainable viticulture.

A key finding of this study was that the suppressive effect on HSVd persisted through three successive subcultures without any additional treatment. The sustained suppression of HSVd in grapevine plantlets that had been previously treated with dsRNAs was confirmed by dPCR analysis. This suggests that RNAi-mediated viroid inhibition may have long-lasting effects, possibly attributable to persistent siRNA accumulation or epigenetic modifications triggered by the initial dsRNA exposure (Ghoshal and Sanfaçon, 2015). The observed long-term persistence highlights the efficacy and practicality of dsRNA-based strategies for viroid management, reducing the need for repeated applications in propagation systems.

To further validate the efficacy of dsRNA treatment, additional experiments were conducted on cucumber plants (Cucumis sativus), a well-established indicator host for HSVd. Following HSVd inoculation, dsRNAs were exogenously applied, and the suppression and absence of HSVd in treated plants were confirmed by RT-PCR analysis. This indicates that dsRNA can effectively inhibit viroid replication irrespective of the host plant. These results emphasize the potential of dsRNA as a broad-spectrum antiviral agent applicable to other susceptible crops and support its practical feasibility for viroid control in diverse agricultural systems.

While our results verified the efficacy of dsRNA application under controlled conditions, the potential for its application in commercial vineyards should also be explored. The direct application of dsRNA as a foliar spray in grapevine propagation systems, as evidenced by our study, may serve as a viable strategy for managing HSVd in agricultural settings. Unlike traditional chemical treatments, dsRNAs can be sprayed directly onto grapevine plants, and preliminary data suggest that it can effectively inhibit viroid replication without negatively affecting plant growth or health. This approach, along with prospective formulation strategies to improve stability and absorption, presents a viable solution for large-scale disease management in vineyards. A recent study demonstrated that trunk injection of dsRNAs in full-sized apple trees enabled systemic delivery through the vascular system, with detectable levels of dsRNAs persisting in the canopy for up to 141 days. This approach effectively addressed environmental degradation issues associated with foliar spray and demonstrated the feasibility of long-term dsRNA retention in woody crops under field conditions, supporting the broader applicability of RNAi-based pest and disease control strategies in tree fruit systems (Wise et al., 2022).

Overall, this study demonstrated the effectiveness of dsRNA in inhibiting HSVd replication, providing compelling molecular evidence supporting RNAi as a viable approach for viroid disease management. The sustained inhibition of HSVd across multiple generations of vegetative propagation further supports the potential of dsRNA application as a long-term disease control strategy. These findings suggest that dsRNA-based strategies could be effectively integrated into grapevine cultivation to reduce HSVd infections. If successfully implemented under field conditions, dsRNA application could facilitate the generation of viroid-free planting material, thereby supporting commercial breeding and advancing sustainable viticulture.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This research was financially supported by Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries (iPET)(320040-05-5-WT011). We would like to thank Professor Jung-Hoon Cho at Chosun University for kindly providing the pL4440 vector and E. coli HT115 (DE3) strain.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-05-2025-0071-Supplementary-Fig.pdf

PPJ-OA-05-2025-0071-Supplementary-Table-1,2,3.pdf

PPJ-OA-05-2025-0071-Supplementary-Table-4.pdf

Fig. 1

Inhibitory effect of hop stunt viroid (HSVd)-specific double-stranded RNAs (dsRNAs) in grapevine plantlets following a single spray application. (A) Reverse transcription polymerase chain reaction analysis of HSVd accumulation in dsRNA-treated grapevine plantlets. (B) Absolute quantification of HSVd copy numbers (copies/μL) using digital PCR. dpt, days post-treatment.

Fig. 2

Inhibitory effect of hop stunt viroid (HSVd)-specific double-stranded RNAs (dsRNAs) in micropropagated grapevine plantlets. (A) Phenotypic comparison of grapevine plantlets exogenously treated with HSVd-specific dsRNAs (bottom) and untreated controls (top) at 28 days post-treatment (dpt). (B) Reverse transcription polymerase chain reaction analysis of HSVd accumulation in dsRNA-treated and control plantlets at 0 and 28 dpt. (C) Absolute quantification of HSVd copy number (copies/μL) by digital PCR, illustrating a reduction in viral load in dsRNA-treated plantlets after 28 days.

Fig. 3

Visualization and uptake of Cy3-labeled hop stunt viroid (HSVd)-specific double-stranded RNAs (dsRNAs) in grapevine plantlets. (A) Electrophoretic validation of Cy3-labeled HSVd-specific dsRNAs. (B) Quantification of Cy3 fluorescence intensity in treated grapevine tissues, indicating dsRNA uptake. Asterisks (***) indicate a statistically significant difference (P < 0.001) between the dsRNA-treated group and the control, as determined by Student’s t-test. Data are presented as mean ± standard deviation (SD).(C) Confocal laser scanning microscopy images illustrating the cellular uptake and distribution of Cy3-dsRNA in grapevine leaves. Fluorescence was observed in mesophyll cells and leaf veins, while no signal was detected in controls.

Fig. 4

Inhibitory effect of hop stunt viroid (HSVd)-specific double-stranded RNAs (dsRNAs) in pot-grown cucumber plants. (A) Phenotypic comparison of cucumber plants at 30 days post inoculation (dpi). HSVd (-): untreated plants; dsRNA: plants treated with HSVd-specific dsRNAs 1 h after HSVd inoculation; HSVd (+): plants inoculated with HSVd inoculum only. (B) Reverse transcription polymerase chain reaction detection of HSVd demonstrating amplification only in HSVd-inoculated plants and an absence of detectable HSVd in dsRNA-treated plants.

Fig. 5

Small interfering RNA (siRNA) profiling in grapevine leaves following hop stunt viroid (HSVd)-specific double-stranded RNAs (dsRNA) treatment. (A) Genome-wide distribution of HSVd-derived siRNAs mapped to the HSVd genome in both sense (positive) and antisense (negative) orientation. (B) Length distribution of siRNA reads in untreated and HSVd-dsRNA-treated grapevine leaves, indicating increased accumulation of 21-24 nt siRNAs upon treatment.

Fig. 6

Sustained suppression of hop stunt viroid (HSVd) across three generations of grapevine plantlets following double-stranded RNA (dsRNA) treatment. (A) Phenotypes of grapevine plantlets successively propagated over three generations without receiving additional treatment after the initial application of HSVd-dsRNAs. Control: plants treated with DEPC-treated water; dsRNA: plants treated with HSVd-dsRNAs. (B) Reverse transcription polymerase chain reaction analysis of HSVd presence in third-generation plantlets. (C) Absolute quantification of HSVd concentration in third-generation plantlets using dPCR.

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