siRNA biogenesis and advances in topically applied dsRNA for controlling virus infections in tomato plants

The study addresses the need for non-transgenic, rapid, and specific antiviral strategies in tomato, a crop significantly affected by viral diseases. RNA interference (RNAi) provides antiviral defense via Dicer-like processing of dsRNA into siRNAs that guide Argonaute-mediated silencing. Building on advances in exogenous dsRNA production and application, the authors aimed to develop and characterize a topical dsRNA method to induce resistance in tomato against RNA viruses (ToMV and PVY) and evaluate its applicability to a DNA begomovirus (ToSRV). They investigated dose dependence, sequence specificity, durability, application methods, systemic transport of dsRNA/siRNA, and siRNA biogenesis by deep sequencing to understand efficacy and mechanistic underpinnings.

Prior work has demonstrated transgenic RNAi (hairpin RNAs) confers virus resistance but raises regulatory and deployment challenges. Exogenously applied dsRNA has emerged as a non-transgenic alternative, with reports of protection against multiple RNA viruses in several hosts using in vitro/in vivo produced dsRNA, crude bacterial extracts, and novel delivery methods (e.g., high-pressure spraying, cell-penetrating peptides, clay nanosheets). Systemic movement of dsRNA and/or siRNA and the roles of DCL and AGO families in antiviral defense have been described, though consistency and durability vary. Evidence for efficacy against DNA viruses is mixed, potentially depending on siRNA size classes (e.g., 24-nt DCL3 products implicated in TGS) and delivery/inoculation conditions.

• Viruses and plants: Tomato cv. Santa Clara grown under controlled conditions; ToMV-BR01 and PVY-To1 inoculated mechanically; ToSRV-1164 (begomovirus) transmitted by Bemisia tabaci MEAM1 (~30 viruliferous whiteflies/plant). Hypersensitive hosts Chenopodium quinoa and Nicotiana glutinosa used for preliminary lesion assays.
• dsRNA design/production: ToMV dsRNA targeting CP (456 bp) and MP (722 bp) generated via in vitro transcription (MEGAscript RNAi Kit); CP dsRNA selected for tomato trials. PVY dsRNA targeted CP (481 bp). ToSRV dsRNA targeted region spanning cp and REn (496 bp) designed via E-RNAi. Final dsRNAs synthesized commercially (AgroRNA).
• Application methods: Mechanical rubbing with carborundum; spray with abrasive; spray without abrasive; root immersion (~3 h). Dose-response tested with ToMV-CP dsRNA at 0, 1, 4, 8, 16, 50, 100, 200, 400 µg/plant. Standardized at 200 µg/plant thereafter. Virus inoculation performed 24 h post-application unless otherwise stated.
• Specificity: Cross-challenge experiments applying PVY-dsRNA prior to PVY or ToMV inoculation and ToMV-dsRNA prior to ToMV or PVY inoculation; infection assessed by indirect-ELISA (ToMV at 7 dpi, PVY at 10 dpi).
• Durability: After ToMV-dsRNA application (day 0), ToMV inoculated at 0–7 days post-treatment (dpt); infection measured at 7 dpi by ELISA.
• Systemic transport of long dsRNA: ToMV-dsRNA applied to one leaf isolated with aluminum foil; treated and upper non-treated leaves sampled at 1, 6 hpt and 1–10 dpt. Total RNA extracted; RT-PCR for dsRNA fragment; chloroplast transcript as endogenous control.
• siRNA HTS: Five treatments—(i) dsRNA+ToMV(+) (infection), (ii) dsRNA+ToMV(-) (protected), (iii) ToMV (infection), (iv) dsRNA only, (v) mock—sampled 6 days after dsRNA application from non-treated leaves. Small RNAs (20–25 nt) sequenced (Illumina HiSeq 2500). Reads adapter-trimmed (BBDuk), mapped simultaneously to tomato genome (GCA_000188115) and ToMV (FN985165) with BWA aln; coverage and size/5′-nt distributions computed (SAMtools, in-house scripts). Biological replicates: three per treatment, each from four plants. Statistical analyses by ANOVA and Tukey tests where applicable.

• Dose dependence: 0–16 µg/plant ToMV-CP dsRNA conferred no protection (100% infection). From 50 µg upward, infection rates decreased: overall 57%→37% infected with increased dsRNA; best at 200 µg (40% infected; ~60% protection) and 400 µg (37% infected; ~63% protection).
• Application method: Mechanical and spray with abrasive yielded highest protection (~57% and ~52% average protection, respectively). Spray without abrasive showed only ~19% protection (not significantly different from control). Root immersion ineffective.
• Symptomatology: Among infected plants, dsRNA-treated plants showed milder symptoms than untreated controls; ELISA-negative plants remained asymptomatic.
• Specificity: PVY-CP dsRNA protected against PVY (~57% protection) but not ToMV (0% protection); ToMV-CP dsRNA protected against ToMV but not PVY, confirming sequence specificity.
• Durability: Maximum protection when virus was co-applied with dsRNA (34% infection). Protection strongest up to 2 dpt; diminished by 3–4 dpt; by day 5 post-application all plants were infected.
• Long dsRNA fate: ToMV dsRNA (456 bp) detectable by RT-PCR in treated leaves up to 10 dpt; not detected in non-treated upper leaves at any timepoint, indicating no systemic movement of long dsRNA.
• siRNA biogenesis and movement: In infected libraries, 34–35% of 20–25 nt reads were ToMV-derived; in mock, dsRNA-only, and dsRNA+ToMV(-) libraries, 96–97% mapped to tomato. Non-treated upper leaves of dsRNA-only plants contained siRNAs mapping to the ToMV CP region, indicating endogenous processing and systemic movement of siRNAs. Size classes predominantly 21- and 22-nt in most treatments; dsRNA-only showed relatively higher 20–22 nt vs 23–25 nt. 5′-U enriched in 21-nt and 5′-A in 22-nt siRNAs; 5′-G least abundant. In protected plants [dsRNA+ToMV(-)], no ToMV-specific siRNAs detected, consistent with absence of viral replication.
• Viral siRNA distribution: In infected plants, siRNAs covered the entire ToMV genome with similar sense/antisense profiles; two hotspots observed within the RNA-dependent RNA polymerase (RDR) ORF. dsRNA pretreatment did not markedly alter siRNA distribution in infections that established.
• Begomovirus test: ToSRV-dsRNA failed to protect tomato under high whitefly inoculation pressure in three independent trials (0/34 protected; all showed symptoms), suggesting limited efficacy against phloem-limited DNA viruses under these conditions.

The findings demonstrate that topically applied dsRNA can induce rapid, sequence-specific, and dose-dependent resistance against RNA viruses (ToMV and PVY) in tomato without genetic modification. Effective protection required sufficiently high dsRNA doses and delivery methods that facilitate cellular entry (abrasive-assisted mechanical application or spraying). Protection was transient, peaking at co-application through 1–2 days post-application, aligning with the observed lack of systemic transport of long dsRNA despite its persistence on treated leaves. Conversely, siRNAs derived from the applied dsRNA were detected systemically, indicating endogenous processing and movement that likely contribute to the protective effect. siRNA profiles (predominantly 21- and 22-nt, with 5′-U/A biases) implicate DCL4/DCL2 and AGO1/AGO2 activities in antiviral defense. The failure to protect against ToSRV highlights potential limitations of this approach against DNA viruses, possibly due to insufficient 24-nt siRNAs (DCL3 products involved in TGS), high inoculum pressure, and/or target selection and inoculation method. Overall, the results support the feasibility of exogenous dsRNA as a practical antiviral tool for RNA viruses, while emphasizing the need to optimize delivery, dosing, and durability for field use.

This study establishes that topical application of dsRNA targeting viral coat protein genes can confer significant, sequence-specific resistance against ToMV and PVY in tomato, with rapid onset and measurable, though transient, protection. It elucidates siRNA biogenesis and systemic movement following dsRNA application and clarifies that long dsRNA remains localized while processed siRNAs move systemically. The work underscores critical parameters—dose, delivery method, and timing—for maximizing efficacy and reveals limitations against a phloem-limited DNA virus (ToSRV). Future research should focus on enhancing delivery and stability (e.g., carriers/encapsulation, high-pressure spraying), extending protection duration, optimizing target regions (including promoting 24-nt siRNAs for DNA viruses), reducing required doses, and validating performance under field conditions and diverse virus-host systems.

• Protection was transient, strongest within 0–2 days post-application and largely lost by 5 days.
• No systemic movement of long dsRNA was detected; efficacy depends on local entry via wounding/abrasive.
• Variable protection rates (~60% at optimal doses) indicate plant-to-plant variability in RNAi responsiveness.
• Root immersion and spraying without abrasive were ineffective, limiting practical delivery options without optimized formulations.
• The approach failed against ToSRV (a phloem-limited DNA virus) under high vector inoculation pressure; low 24-nt siRNA production and target/inoculation method may constrain efficacy.
• Results may depend on virus strain, host genotype, dsRNA purity, and environmental conditions, which were controlled in growth chambers and may differ in field settings.