Spray-induced Gene Silencing Research Articles

Double-stranded RNAs targeting Sphaerulina musiva silence DCL but not CYP51 in vitro, and fail to confer resistance to canker in transgenic poplar

Host-induced gene silencing (HIGS) is a common method for engineering plant protection against pathogens, although success requires double-stranded (dsRNA) uptake mechanisms that may not be present in all fungi. We explored HIGS in transgenic poplar to study and control Sphaerulina musiva, the cause of Septoria stem canker disease. HIGS transgenic poplars expressing dsRNA that targeted either or both S. musiva CYP51 and DCL were developed and screened for resistance to stem canker disease in two greenhouse inoculation trials. While differences in resistance between transgenic lines and wild-type controls were not detected, there was a correlation between greenhouse-expressed disease resistance and transgene expression among HIGS lines targeting S. musiva DCL. To evaluate the likelihood that HIGS or spray-induced gene silencing might be effective under some conditions, concurrent with greenhouse screening we studied: 1) S. musiva’s capacity for uptake of environmental dsRNA; 2) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and 3) persistence of dsRNA in culture. Uptake of fluorescently tagged dsRNA was not detected with confocal imaging. In dsRNA-treated cultures, fungal growth inhibition was not detected, and RNA was rapidly degraded. Of the five target transcripts tested after dsRNA treatment, only DCL1 had reduced expression. Knockdown of DCL1 along with the enhanced resistance among high-expressing HIGS events targeting DCL suggests some HIGS may have been observed. Further determination of the factors limiting dsRNA uptake by S. musiva are needed to determine if HIGS can be an effective technology for limiting stem canker.

Extracellular Vesicles Mimetic Design of Membrane Chimeric Nanovesicles for dsRNA Delivery in Spray-Induced Gene Silencing for Crop Protection.

Spray-induced gene silencing (SIGS) presents a promising RNA interference (RNAi)-based crop protection strategy against eukaryotic phytopathogens. However, the application of SIGS faces challenges, such as the limited uptake of dsRNA by certain pathogens and the instability of dsRNA in the environment. This study introduces innovative biomimetic nanovesicles, called extracellular vesicle (EV) mimetic chimeric nanovesicles (ECNs), assembled from tomato leaf cell membranes and cationic sterosomes via the freeze-thaw method. Similar to the function of EVs in nucleic acid transport between cells, ECNs serve as a hybrid nanosystem to overcome the challenge of delivering exogenous dsRNA in Phytophthora infestans. When applied to SIGS, the superiority of ECNs in crop protection becomes more apparent, including high loading and protection of dsRNA, improved biosafety, and efficient internalization into pathogen and plant cells, all of which significantly enhance the efficacy of RNAi in preventing early infection of P. infestans to susceptible tomato plants. This study demonstrates that ECNs are promising RNA delivery vehicles and will promote the use of SIGS-based RNA pesticides in sustainable agricultural production.

Cross-Kingdom RNA Transport Based on Extracellular Vesicles Provides Innovative Tools for Plant Protection.

RNA interference (RNAi) shows great potential in plant defense against pathogens through RNA-mediated sequence-specific gene silencing. Among RNAi-based plant protection strategies, spray-induced gene silencing (SIGS) is considered a more promising approach because it utilizes the transfer of exogenous RNA between plants and microbes to silence target pathogen genes. The application of nanovesicles significantly enhances RNA stability and delivery efficiency, thereby improving the effectiveness of SIGS and further enhancing plant resistance to diseases and pathogens. This review explores the role of RNAi in plant protection, focusing on the cross-kingdom transport of small RNAs (sRNAs) via extracellular vesicles. It also explores the potential of nanotechnology to further optimize RNA-based plant protection, offering innovative tools and methods in modern plant biotechnology.

Suppression of Thrips palmi population by spray-on application of dsRNA targeting V-ATPase-B

The RNA interference (RNAi)-based gene silencing technique has enormous potential as a non-chemical and eco-friendly alternative to hazardous pesticides. This study reports a spray-induced gene silencing (SIGS) approach for managing Thrips palmi by lowering survival and offspring development. Vacuolar ATP synthases (V-ATPases) are responsible for survival, egg-laying, and viability of eggs in insects. In the current study, T. palmi V-ATPase-B was targeted to suppress the pest population by spray-on application of double-stranded RNA (dsRNA). Silencing of V-ATPase-B was first validated by oral administration of dsV-ATPase-B. The expression of V-ATPase-B was reduced by 5.40-fold post-dsRNA feeding leading to increased mortality (57.03 %) and reduced reproductive fitness (67.73 %). Spray-on application of naked dsV-ATPase-B at concentrations of 3.0 μg/mL and 5.0 μg/mL effectively suppressed the population by 30.00 % and 43.33 %, respectively. The expression of the target gene was downregulated by up to 4.24-fold. Two consecutive sprays at a concentration of 5.0 μg/mL provided substantial protection against the fresh release of T. palmi for up to 10 days. The spray-on application of dsV-ATPase-B would be an eco-friendly alternative for managing T. palmi populations thereby reducing crop damage and limiting the spread of orthotospoviruses.

Regulation of Anthocyanin Accumulation in Tomato Solanum lycopersicum L. by Exogenous Synthetic dsRNA Targeting Different Regions of SlTRY Gene.

RNA interference (RNAi) is a regulatory and protective mechanism that plays a crucial role in the growth, development, and control of plant responses to pathogens and abiotic stresses. In spray-induced gene silencing (SIGS), exogenous double-stranded RNAs (dsRNA) are used to efficiently regulate target genes via plant surface treatment. In this study, we aimed to evaluate the effect of specific exogenous dsRNAs on silencing different regions (promoter, protein-coding and intron) of the target SlTRY tomato gene, encoding an R3-type MYB repressor of anthocyanin biosynthesis. We also assessed the impact of targeting different SlTRY regions on the expression of genes involved in anthocyanin and flavonoid biosynthesis. This study demonstrated the critical importance of selecting the appropriate gene target region for dsRNA action. The highest inhibition of the SlTRY gene expression and activation of anthocyanin biosynthesis was achieved by dsRNA complementary to the protein-coding region of SlTRY gene, compared with dsRNAs targeting the SlTRY promoter or intron regions. Silencing the SlTRY gene increased the content of anthocyanins and boosted levels of other substances in the phenylpropanoid pathway, such as caffeoyl putrescine, chlorogenic acid, ferulic acid glucoside, feruloyl quinic acid, and rutin. This study is the first to examine the effects of four different dsRNAs targeting various regions of the SlTRY gene, an important negative regulator of anthocyanin biosynthesis.

Spray-induced gene silencing (SIGS) as a tool for the management of Pine Pitch Canker forest disease.

Global change is exacerbating the prevalence of plant diseases caused by pathogenic fungi in forests worldwide. The conventional use of chemical fungicides, which is commonplace in agricultural settings, is not sanctioned for application in forest ecosystems, so novel control strategies are imperative. SIGS (Spray-Induced Gene Silencing) is a promising approach that can modulate the expression of target genes in eukaryotes in response to double-stranded RNA (dsRNA) present in the environment that triggers the RNA interference (RNAi) mechanism. SIGS exhibited notable success in reducing virulence when deployed against some crop fungal pathogens, such as Fusarium graminearum, Botrytis cinerea and Sclerotinia sclerotiorum, among others. However, there is a conspicuous dearth of studies evaluating the applicability of SIGS for managing forest pathogens. This research aimed to determine whether SIGS could be used to control Fusarium circinatum, a widely impactful forest pathogen that causes Pine Pitch Canker disease. Through a bacterial synthesis, we produced dsRNA molecules to target fungal essential genes involved to vesicle trafficking (Vps51, DCTN1, and SAC1), signal transduction (Pp2a, Sit4, Ppg1, and Tap42), and cell wall biogenesis (Chs1, Chs2, Chs3b, Gls1) metabolic pathways. We confirmed that F. circinatum is able to uptake externally applied dsRNA, triggering an inhibition of the pathogen's virulence. Furthermore, this study pioneers the demonstration that recurrent applications of dsRNAs in SIGS are more effective in protecting plants than single applications. Therefore, SIGS emerges as an effective and sustainable approach for managing plant pathogens, showcasing its efficacy in controlling a globally significant forest pathogen subject to quarantine measures.

The genome sequencing and comparative genomics analysis of Rhizoctonia solani reveals a novel effector family owning a uinque domain in Basidiomycetes

Rhizoctonia solani is a soil-borne pathogen with 14 anastomosis groups (AGs), and different subgroups are genetically diverse. However, the genetic factors contributing to the pathogenicity of the fungus have not been well characterized. In this study, the genome of R. solani AG1-ZJ was sequenced. As the result, a 41.57 Mb draft genome containing 12,197 putative coding genes was obtained. Comparative genomic analysis of 11 different AGs revealed conservation and unique characteristics between the AGs. Furthermore, a novel effector family containing a 68 amino acid conserved domain unique in basidiomycetous fungi was characterized. Two effectors containing the conserved domain in AG4-JY were identified, and named as RsUEB1 and RsUEB2. Furthermore, the spray-induced gene silencing strategy was used to generate a dsRNA capable of silencing the conserved domain sequence of RsUEB1 and RsUEB2. This dsRNA can significantly reduce the expression of RsUEB1 and RsUEB2 and the pathogenicity of AG4-JY on foxtail millet, maize, rice and wheat. In conclusion, this study provides significant insights into the pathogenicity mechanisms of R. solani. The identification of the conserved domain and the successful use of dsRNA silencing of the gene containing the conserved domain will offer a new strategy for controlling sheath blight in cereal crops.

Development of an RNA Nanostructure for Effective Botrytis cinerea Control through Spray-Induced Gene Silencing without an Extra Nanocarrier.

Spray-induced gene silencing represents an eco-friendly approach for crop protection through the use of double-stranded RNA (dsRNA) to activate the RNA interference (RNAi) pathway, thereby silencing crucial genes in pathogens. The major challenges associated with dsRNA are its limited stability and poor cellular uptake, necessitating repeated applications for effective crop protection. In this study, RNA nanoparticles (NPs) were proposed as effectors in plants and pathogens by inducing the RNAi pathway and silencing gene expression. RNA structural motifs, such as hairpin-loop, kissing-loop, and tetra-U motifs, were used to link multiple siRNAs into a long, single-stranded RNA (lssRNA). The lssRNA, synthesized in Escherichia coli, self-assembled into stable RNA nanostructures via local base pairing. Comparative analyses between dsRNA and RNA NPs revealed that the latter displayed superior efficacy in inhibiting spore germination and mycelial growth of Botrytis cinerea. Moreover, RNA NPs had a more robust protective effect on plants against B. cinerea than did dsRNA. In addition, RNA squares are processed into expected siRNA in plants, thereby inhibiting the expression of the target gene. These findings suggest the potential of RNA NPs for use in plant disease control by providing a more efficient and specific alternative to dsRNA without requiring nanocarriers.

The critical roles of the Zn2Cys6 transcription factor Fp487 in the development and virulence of Fusarium pseudograminearum: A potential target for Fusarium crown rot control

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum poses a significant threat to wheat production in the Huang-Huai-Hai region of China. However, the pathogenic mechanism of F. pseudograminearum is still poorly understood. Zn2Cys6 transcription factors, which are exclusive to fungi, play pivotal roles in regulating fungal development, drug resistance, pathogenicity, and secondary metabolism. In this study, we present the functional characterization of a Zn2Cys6 transcription factor F. pseudograminearum, designated Fp487. In F. pseudograminearum, Fp487 is shown to be required for mycelial growth through gene knockout and phenotypic analyses. Compared with wild-type CF14047, the ∆Fp487 mutant displayed a slight reduction in growth rate but a significant decrease in conidiogenesis, pathogenicity and 3-acetyl-deoxynivalenol (3AcDON) production. Moreover, the mutant exhibited heightened sensitivity to oxidative and cytomembrane stress. Furthermore, we synthesized dsRNA from the Fp487 gene in vitro, resulting in a reduction in the growth rate of F. pseudograminearum and its virulence on barley leaves through spray-induced gene silencing (SIGS). Notably, this study makes the first instance of inducing the expression of abundant dsRNA from F. pseudograminearum by engineering the Escherichia coli strain HT115 (DE3) and utilizing the SIGS technique to evaluate the virulence effect of dsRNA on F. pseudograminearum. In conclusion, our findings revealed the crucial role of Fp487 in regulating pathogenicity, stress responses, DON production, and conidiogenesis in F. pseudograminearum. Furthermore, Fp487 is a potential RNAi-based target for FCR control.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against thebroad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Plant-derived artificial miRNA effectively reduced the proliferation of aphid (Aphidoidea) through spray-induced gene silencing.

Aphids (Hemiptera: Aphididae) are notorious sap-sucking insects that rampantly threaten agricultural production worldwide. Current management against aphids in the field heavily relies on chemical pesticides, which makes economical and eco-friendly methods urgently needed. Spray-induced gene silencing (SIGS) offers a powerful and precise approach to pest management. However, the high costs and instability of double-stranded RNA (dsRNA) regulators applied for downstream RNA interference (RNAi) still limit this strategy. It remains uncertain if RNAi regulators applied in SIGS could extend to small RNA (sRNA), especially miRNA. We chose two sRNA sequences, miR-9b and miR-VgR, whose corresponding targets ABCG4 and VgR are both essential for aphid growth and development. The efficacy of these sequences was initially verified by chemically synthetic single-stranded RNA (syn-ssRNA). Through spray treatment, we observed a significantly decreased survival number and increased abnormality rate of green peach aphids fed on the host under laboratory conditions. Based on our previous study, we generated transgenic plants expressing artificial miR-9b (amiR-9b) and miR-VgR (amiR-VgR). Remarkably, plant-derived amiRNA exerted potent and long-lasting inhibitory efficacy with merely one percent concentration of chemical synthetics. Notably, the simultaneous application of amiR-9b and amiR-VgR exhibited superior inhibitory efficacy. We explored the potential use of sRNA-based biopesticide through SIGS while investigating the dosage requirements. To optimize this strategy, the utilization of plant-derived amiRNA was proposed. The results suggested that attributed to stability and durability, deploying amiRNA in pest management is a potential and promising solution for the field application. © 2024 Society of Chemical Industry.

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants.

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

Advances in the mechanisms and applications of RNA silencing in crop protection.

RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes, which plays vital roles in plant development and response to biotic and abiotic stresses. The discovery of trans-kingdom RNAi and interspecies RNAi provides a theoretical basis for exploiting RNAi-based crop protection strategies. Here, we summarize the canonical RNAi mechanisms in plants and review representative studies associated with plant-pathogen interactions. Meanwhile, we also elaborate upon the principles of host-induced gene silencing, spray-induced gene silencing and microbe-induced gene silencing, and discuss their applications in crop protection, thereby providing help to establish novel RNAi-based crop protection strategies.

The development of an egg-soaking method for delivering dsRNAs into spider mites

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Silence of five F. graminearum genes in wheat host confers resistance to Fusarium head blight

Fusarium head blight (FHB), mainly caused by fungus Fusarium graminearum (F. graminearum), is a devastating wheat disease worldwide, leading to reduced yield production and compromised grain quality due to contamination by mycotoxins, such as deoxynivalenol (DON). Manipulating the specific gene expression in microorganisms through RNA interference (RNAi) presents an opportunity for new-generation double-stranded RNA (dsRNA)-based formulations to combat a large number of plant diseases. Here, we applied both spray-induced gene silencing (SIGS) and host-induced gene silencing (HIGS) to target five virulence-related and DON-synthesized genes in F. graminearum, including protein kinase gene Gpmk1, zinc finger protein gene FgChy1, transcription factor FgSR, DON synthesis gene TRI5 and the cell-end marker protein gene FgTeaA, aiming to effectively control FHB in wheat. Direct spraying of individual or combined siRNAs (small interfering RNA) from the fungus showed reduced expression of target genes and suppressed pathogenic symptoms during F. graminearum infection in wheat leaves, with the combination of all five siRNAs demonstrating superior resistance. Furthermore, we generated transgenic wheat lines expressing chimeric RNAi cassettes targeting these five genes, and two independent lines exhibited strong resistance to FHB and Fusarium crown rot, and the reduced DON accumulation. Notably, the HIGS transgenic lines did not adversely impact plant growth and yield traits. Collectively, our findings support that SIGS and HIGS represent effective strategies targeting key pathogenic genes for bolstering disease resistance in crops.

Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

Spray-induced and nanocarrier-delivered gene silencing system targeting juvenile hormone receptor components: potential application as fertility inhibitors for Adelphocoris suturalis management.

Adelphocoris suturalis is a destructive pest that attacks > 270 plants, including cotton, maize, soybean, and fruit trees. Adelphocoris suturalis can cause tremendous crop losses when the density exceeds economic thresholds, but because it can be both phytophagous and zoophytophagous it can serve as a natural enemy of other pests when the density is below the economic threshold. Effective control of its population is beneficial for maximizing yield and profits. RNA interference (RNAi) has potential to be a viable alternative to conventional pesticide-based pest management, but the lack of efficient double-stranded RNA (dsRNA) delivery systems and candidate genes are currently limiting factors for field applications. In this study, RNAi of juvenile hormone (JH) receptor components methoprene-tolerant (Met)/Taiman (Tai) in Adelphocoris suturalis reduced fertility. Based on this reproductive role, we targeted Adelphocoris suturalis Met and Tai for knockdown by coupling nanomaterial-dsRNA complexes with a transdermal spray delivery system. Within 12 h of adult emergence, females were sprayed with star polycation (SPc)-dsRNA formulations and the RNAi effects were assessed over time. RNAi knockdown efficiencies of 39-58% were observed at 5 days post-treatment and abnormal ovarian development was apparent by 10 days post-treatment. Our results show that spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system targeting JH signal genes effectively impaired oviposition, thus developing a novel RNA fertility inhibitor to control Adelphocoris suturalis populations. These results give new perspective on pest management and suggest broad prospects for field applications. © 2024 Society of Chemical Industry.

Dissecting Diagnostic and Management Strategies for Plant Viral Diseases: What Next?

Recent advancements in molecular biology have revolutionized plant disease diagnosis and management. This review focuses on disease diagnosis through serological techniques, isothermal amplification methods, CRISPR-based approaches, and management strategies using RNA-based methods. Exploring high-throughput sequencing and RNA interference (RNAi) technologies like host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), this review delves into their potential. Despite the precision offered by RNAi in pest and pathogen management, challenges such as off-target effects and efficient dsRNA delivery persist. This review discusses the significance of these strategies in preventing aphid-mediated plant virus transmission, emphasizing the crucial role of meticulous dsRNA design for effective viral RNA targeting while minimizing harm to plant RNA. Despite acknowledged challenges, including off-target effects and delivery issues, this review underscores the transformative potential of RNA-based strategies in agriculture. Envisaging reduced pesticide dependency and enhanced productivity, these strategies stand as key players in the future of sustainable agriculture.

Double-Stranded RNA Targeting White Mold Sclerotinia sclerotiorum Argonaute 2 for Disease Control via Spray-Induced Gene Silencing.

Sclerotinia sclerotiorum, the causal agent of white mold infection, is a cosmopolitan fungal pathogen that causes major yield losses in many economically important crops. Spray-induced gene silencing has recently been shown to be a promising alternative method for controlling plant diseases. Based on our prior research, we focused on developing a spray-induced gene silencing approach to control white mold by silencing S. sclerotiorum argonaute 2 (SsAgo2), a crucial part of the fungal small RNA pathway. We compared the lesion size as a result of targeting each ∼500-bp segment of SsAgo2 from the 5' to the 3' end and found that targeting the PIWI/RNaseH domain of SsAgo2 is most effective. External application of double-stranded RNA (dsRNA)-suppressed white mold infection using either in vitro or in vivo transcripts was determined at the rate of 800 ng/0.2 cm2 area with a downregulation of SsAgo2 from infected leaf tissue confirmed by RT-qPCR. Furthermore, magnesium/iron-layered double hydroxide nanosheets loaded with in vitro- and in vivo-transcribed dsRNA segments significantly reduced the rate of S. sclerotiorum lesion expansion. In vivo-produced dsRNA targeting the PIWI/RNaseH domain of the SsAgo2 transcript showed increased efficacy in reducing the white mold symptoms of S. sclerotiorum when combined with layered double hydroxide nanosheets. This approach is promising to produce a large scale of dsRNA that can be deployed as an environmentally friendly fungicide to manage white mold infections in the field.

RNA Interference-Screening of Potentially Lethal Gene Targets in the White-Backed Planthopper Sogatella furcifera via a Spray-Induced and Nanocarrier-Delivered Gene Silencing System.

RNA interference (RNAi) is a widespread post-transcriptional silencing mechanism that targets homologous mRNA sequences for specific degradation. An RNAi-based pest management strategy is target-specific and considered a sustainable biopesticide. However, the specific genes targeted and the efficiency of the delivery methods can vary widely across species. In this study, a spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system that incorporated gene-specific dsRNAs targeting conserved genes was used to evaluate phenotypic effects in white-backed planthopper (WBPH). At 2 days postspraying, transcript levels for all target genes were significantly reduced and knockdown of two gene orthologs, hsc70-3 and PP-α, resulted in an elevated mortality (>60%) and impaired ecdysis. These results highlight the utility of the SI-NDGS system for identifying genes involved in WBPH growth and development that could be potentially exploitable as high mortality target genes to develop an alternative method for WBPH control.