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

Tomato production faces significant losses due to viral diseases. Transgenic approaches using RNA interference (RNAi) have shown promise in conferring virus resistance by expressing viral dsRNA which is processed into siRNAs. These siRNAs, along with microRNAs (miRNAs), are loaded into Argonaute (AGO) proteins and incorporated into the RNA-induced silencing complex (RISC), leading to post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS). This process requires host RNA-dependent RNA polymerases (RDRs) to amplify the antiviral response, and the subsequent systemic movement of siRNAs is crucial for whole-plant protection. Recent advances in dsRNA production have enabled the exploration of topical dsRNA application as a non-transgenic alternative to circumvent transformation challenges and biosafety concerns. Several studies have demonstrated that exogenous dsRNA can induce protection against various plant viruses. This research aimed to develop a dsRNA topical application method for controlling tomato virus diseases, evaluating dose-response, specificity, durability, application methods, systemic dsRNA transport, and siRNA biogenesis through deep sequencing.

The use of RNAi for conferring virus resistance in plants has been extensively studied. Transgenic approaches, involving the expression of double-stranded RNA (dsRNA) homologous to viral sequences, have demonstrated efficacy. However, the complexity and regulatory hurdles associated with transgenic approaches have motivated the exploration of non-transgenic strategies. Topical application of exogenous dsRNA, produced in vitro or in vivo, has emerged as an attractive alternative. Numerous studies have shown the successful application of this method against various plant viruses, including members of Tobamovirus, Potyvirus, Alfamovirus, and Potexvirus. These studies, however, vary significantly in the concentration and type of dsRNA applied and the delivery method used. The present study builds on these previous findings by focusing on the optimization of topical dsRNA application for virus control in tomato plants, with a particular emphasis on understanding the mechanisms of siRNA biogenesis and transport.

Tomato plants (cv. Santa Clara) were used in this study, along with *Chenopodium quinoa* and *Nicotiana glutinosa* for preliminary assays. dsRNA molecules targeting the cp and mp genes of ToMV were synthesized in vitro using the MEGAscript RNAi Kit. For PVY and ToSRV, dsRNA was designed using the E-RNAi tool and synthesized by AgroRNA. Initial experiments tested dsRNA efficacy against ToMV in *C. quinoa* and *N. glutinosa*, assessing lesion reduction. Dose-response studies in tomato plants involved applying various concentrations (0-400 µg/plant) of ToMV-dsRNA, followed by ToMV inoculation 24 hours later. Infection rates were determined at 7 dpi using indirect-ELISA. Different application methods (mechanical, spraying with/without abrasive, root immersion) were compared. Specificity was assessed by testing PVY-dsRNA against PVY and ToMV infections. Durability of protection was evaluated by inoculating ToMV at different time points (0-7 days) post-dsRNA application. Systemic dsRNA transport was monitored by RT-PCR in treated and untreated leaves at various time points (1 and 6 h, 1-10 days). Finally, siRNA populations were analyzed by high-throughput sequencing (HTS) in five treatments: (i) dsRNA+ToMV(+) (infected), (ii) dsRNA+ToMV(-) (protected), (iii) ToMV, (iv) dsRNA, and (v) mock. Illumina sequencing of 15 cDNA libraries was performed, with reads mapped to ToMV and tomato genome sequences. Data analysis included determination of siRNA size class profiles and 5'-nucleotide identities, along with single-nucleotide resolution maps.

Topical application of ToMV-dsRNA resulted in a dose-dependent and sequence-specific reduction in ToMV infection rates in tomato plants. The highest protection (60-63%) was observed with 200 and 400 µg/plant doses. Similar levels of protection were achieved against PVY using PVY-dsRNA. Mechanical application or spraying with abrasive proved the most effective dsRNA delivery methods. Protection was transient, lasting up to 4 days. Long dsRNA persisted in treated leaves for at least 10 days, but systemic movement was not detected. However, dsRNA-derived siRNAs (mainly 21- and 22-nt) were found in untreated leaves, indicating endogenous processing and transport. In contrast, no protection was observed against ToSRV, a phloem-limited DNA virus. Deep sequencing revealed that in protected plants, siRNA levels were similar to mock-treated plants, while plants treated with dsRNA alone exhibited higher siRNA levels, specifically in the 20-23 nt range. The analysis of siRNA size class profiles and 5'-nucleotide identities showed predominantly 21- and 22-nt siRNAs, with a prevalence of 5'U and 5'A, suggesting the involvement of AGO1 and AGO2. Two hotspots of sense and antisense siRNAs were identified in the RNA polymerase ORF of ToMV in infected plants.

This study demonstrates the feasibility of using topically applied dsRNA to control RNA viruses in tomato plants. The observed dose-dependent and sequence-specific protection indicates that the exogenous dsRNA is processed by the plant's endogenous RNAi machinery. The transient nature of protection suggests the need for strategies to enhance dsRNA persistence and/or amplify the RNAi response. The lack of protection against ToSRV highlights a potential limitation; the method may not be effective against all types of viruses. The deep sequencing data provide valuable insights into siRNA biogenesis and their role in antiviral defense. The preferential generation of 21- and 22-nt siRNAs, coupled with the 5'-nucleotide preferences, suggests the involvement of specific DCL and AGO proteins in the antiviral response. The identified siRNA hotspots within the viral genome may offer clues for optimizing dsRNA design for enhanced efficacy.

This research successfully demonstrated that topical application of dsRNA can effectively induce resistance against RNA viruses in tomato plants. The findings emphasize the potential of this non-transgenic approach for controlling viral diseases. Further research should focus on strategies to improve the durability of protection, such as optimizing dsRNA delivery methods and exploring the role of different RNAi components in determining the success of virus resistance. The identification of optimal target sequences within the viral genome warrants further investigation. Investigating the efficacy of this method against other plant viruses and in field settings is crucial for translating these findings into practical applications.

The study's primary limitation is the transient nature of the observed protection. While this approach is effective against RNA viruses, it demonstrates limited efficacy against phloem-limited DNA viruses like ToSRV. This necessitates further research into alternative targeting strategies and dsRNA delivery methods for DNA virus control. The study was conducted under controlled environmental conditions, and further investigation under field conditions is needed to validate the findings and determine their practicality. The variability observed in siRNA levels among replicates warrants further investigation to understand the factors contributing to the heterogeneity of the RNAi response.