Knockout of SlDCL2b attenuates the resistance of tomato to potato spindle tuber viroid infection.

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.

Near-infrared light and PIF4 promote plant antiviral defense by enhancing RNA interference.

The molecular mechanism underlying phototherapy and light treatment, which utilize various wavelength spectra of light, including near-infrared (NIR), to cure human and plant diseases, is obscure. Here we revealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-activated RNA interference (RNAi) in plants. PIF4, a central transcription factor involved in light signaling, accumulates to high levels under NIR light in plants. PIF4 directly induces the transcription of two essential components of RNAi, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and ARGONAUTE 1 (AGO1), which play important roles in resistance to both DNA and RNA viruses. Moreover, the pathogenic determinant ßC1 protein, which is evolutionarily conserved and encoded by betasatellites, interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization. These findings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the exploration of NIR antiviral treatment.

Larvae of Colorado potato beetle (Leptinotarsa decemlineata Say) resistant to double-stranded RNA (dsRNA) remain susceptible to small-molecule pesticides.

BACKGROUND: Implementation of resistance management tools is crucial for the continued efficacy of insect control technologies. An important aspect of insect resistance management (IRM) is the combined or sequential use of different modes-of-action to reduce selection pressure and delay evolution of resistance. This is especially important for insect pests with established ability to develop resistance to insecticides, such as the Colorado potato beetle (Leptinotarsa decemlineata, CPB). A new class of insecticides, based on double-stranded RNA (dsRNA) activating the gene silencing RNA-interference (RNAi) pathway, are currently under review for regulatory approval and commercial use in the USA against CPB. However, there is no information available on the potential for cross-resistance between RNAi insecticides and other classes of insecticides used against CPB. Herein, we aim to fill this knowledge gap by capitalizing on the availability of a CPB strain highly resistant to dsRNAs and test its susceptibility to diverse small-molecule insecticide classes compared to reference dsRNA-susceptible CPB strains. RESULTS: Differences in activity were observed among the four insecticides tested, with abamectin demonstrating highest activity against all three strains of CPB. However, no differences were observed among the dsRNA-resistant and susceptible CPB strains for any of the tested compounds. Overall, these results demonstrate lack of cross-resistance to commonly used chemical insecticides in the dsRNA-resistant strain of CPB. CONCLUSION: These data support the use of these different insecticide classes along with RNAi-based insecticides as part of an effective insect resistance management framework aimed at delaying resistance in CPB. © 2023 Society of Chemical Industry.

A Non-Canonical Pathway Induced by Externally Applied Virus-Specific dsRNA in Potato Plants.

The external application of double-stranded RNA (dsRNA) has recently been developed as a non-transgenic approach for crop protection against pests and pathogens. This novel and emerging approach has come to prominence due to its safety and environmental benefits. It is generally assumed that the mechanism of dsRNA-mediated antivirus RNA silencing is similar to that of natural RNA interference (RNAi)-based defence against RNA-containing viruses. There is, however, no direct evidence to support this idea. Here, we provide data on the high-throughput sequencing (HTS) analysis of small non-coding RNAs (sRNA) as hallmarks of RNAi induced by infection with the RNA-containing potato virus Y (PVY) and also by exogenous application of dsRNA which corresponds to a fragment of the PVY genome. Intriguingly, in contrast to PVY-induced production of discrete 21 and 22 nt sRNA species, the externally administered PVY dsRNA fragment led to generation of a non-canonical pool of sRNAs, which were present as ladders of ~18-30 nt in length; suggestive of an unexpected sRNA biogenesis pathway. Interestingly, these non-canonical sRNAs are unable to move systemically and also do not induce transitive amplification. These findings may have significant implications for further developments in dsRNA-mediated crop protection.

Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

A holistic analysis of the intrinsic and delivery-mediated toxicity of siRNA therapeutics.

Small interfering RNAs (siRNAs) are among the most promising therapeutic platforms in many life-threatening diseases. Owing to the significant advances in siRNA design, many challenges in the stability, specificity and delivery of siRNA have been addressed. However, safety concerns and dose-limiting toxicities still stand among the reasons for the failure of clinical trials of potent siRNA therapies, calling for a need of more comprehensive understanding of their potential mechanisms of toxicity. This review delves into the intrinsic and delivery related toxicity mechanisms of siRNA drugs and takes a holistic look at the safety failure of the clinical trials to identify the underlying causes of toxicity. In the end, the current challenges, and potential solutions for the safety assessment and high throughput screening of investigational siRNA and delivery systems as well as considerations for design strategies of safer siRNA therapeutics are outlined.

Inefficient uptake of small interfering RNAs is responsible for their inability to trigger RNA interference in Colorado potato beetle cells.

There has been limited success in the usage of exogenous small interference RNA (siRNA) or small hairpin RNA (shRNA) to trigger RNA interference (RNAi) in insects. Instead, long double-stranded RNAs (dsRNA) are used to induce knockdown of target genes in insects. Here, we compared the potency of si/sh RNAs and dsRNA in Colorado potato beetle (CPB) cells. CPB cells showed highly efficient RNAi response to dsRNA. However, si/sh RNAs were inefficient in triggering RNAi in CPB cells. Confocal microscopy observations of Cy3 labeled-si/sh RNA cellular uptake revealed reduced si/sh RNA uptake compared to dsRNA. si/sh RNAs were stable in the conditioned media of CPB cells. Although in a small amount, when internalized by CPB cells, the si/sh RNAs were processed by the Dicer enzyme. Lipid-mediated transfection and chimeric dsRNA approaches were used to improve the delivery of si/sh RNAs. Our results suggest that the uptake of si/sh RNAs is inefficient in CPB cells, resulting in ineffective RNAi response. However, with the help of effective delivery methods, si/sh RNA could be a useful option for developing target-specific RNAi-mediated biopesticides.

Lack of fitness costs in dsRNA-resistant Leptinotarsa decemlineata ([Coleoptera]: [Chrysomelidae]).

The Colorado potato beetle, Leptinotarsa decemlineata (Say) ([Coleoptera]: [Chrysomelidae]), is the most important defoliator of solanaceous plants worldwide. This insect displays a notorious ability in adapting to biological and synthetic insecticides, although in some cases this adaptation carries relevant fitness costs. Insecticidal gene silencing by RNA interference is a novel mode of action pesticide against L. decemlineata that is activated by ingestion of a double stranded RNA (dsRNA) targeting a vital L. decemlineata gene. We previously reported laboratory selection of a > 11,000-fold resistant strain of L. decemlineata to a dsRNA delivered topically to potato leaves. In this work, we tested the existence of fitness costs in this dsRNA-resistant colony by comparing biological parameters to the parental strain and an additional susceptible reference strain. Biological parameters included length of egg incubation period, number of eggs per clutch, egg viability, larval viability, length of larval and pupal periods, adult emergence, number of eggs laid per day, sex ratio, and adult longevity. Comparisons between the 3 beetle strains detected no fitness costs associated with resistance to dsRNA. This information is important to guide effective insect resistance management plans for dsRNA insecticides against L. decemlineata applied topically to potato leaves.

Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Is withdrawal of nocturnal hyperhydration possible in children with primary hyperoxaluria treated with RNAi?

Primary hyperoxaluria type 1 is a rare genetic disorder caused by bi-allelic pathogenic variants in the AGXT gene leading to an overproduction of oxalate which accumulates in the kidneys in the form of calcium oxalate crystals. Thus, patients may present with recurrent nephrocalcinosis and lithiasis, with progressive impairment of the  renal function and eventually kidney failure.  There is no specific treatment besides liver-kidney transplantation, and pre-transplantation management by 24 h-hyperhydration, crystallisation inhibitors and high-dose pyridoxine has a high negative impact on quality of life, especially because of the discomfort due to nocturnal hyperhydration. Since 2020, lumasiran, an RNA-interfering therapy, has been approved for the treatment of primary hyperoxaluria type 1 in adults and children. However, to date, there are no recommendations regarding the discontinuation of other supportive measures during RNAi therapy. In this report, we present two patients with primary hyperoxaluria type 1 who were treated with lumasiran and stopped nocturnal hyperhydration with positive outcomes, i.e. normal urinary oxalate, absence of crystalluria, stable kidney function and improved well-being. These data suggest that discontinuing nocturnal hydration may be safe in children responding to lumasiran, and may have a positive impact on their quality of life. Additional data are needed to update treatment recommendations.

Identification of leaf chloroplast-specific promoter to efficiently control of Colorado potato beetle with reduced dsRNA accumulation in potato tubers.

BACKGROUND: By expressing double-stranded RNA (dsRNA) in potato plastids targeting the ß-Actin (ACT) gene of the Colorado potato beetle (CPB), transplastomic plants can trigger the beetle's RNA interference response to kill the CPB larvae. High expression of dsACT driven by rrn16 promoter (Prrn) in the leaf chloroplasts of transplastomic plants confers strong resistance to CPB. However, there are still residual amounts of dsRNA in the tubers, which are unnecessary for CPB control and may raise a potential food exposure issue. RESULTS: In order to reduce dsRNA accumulation in the tubers while maintaining stable resistance to CPB, we selected two promoters (PrbcL and PpsbD) from potato plastid-encoded rbcL and psbD genes and compared their activities with Prrn promoter for dsRNA synthesis in the leaf chloroplasts and tuber amyloplasts. We found that the dsACT accumulation levels in leaves of transplastomic plants St-PrbcL-ACT and St-PpsbD-ACT were significantly reduced when compared to St-Prrn-ACT, but they still maintained high resistance to CPB. By contrast, a few amounts of dsACT were still accumulated in the tubers of St-PrbcL-ACT, whereas no dsACT accumulation in tubers was detectable in St-PpsbD-ACT. CONCLUSION: We identified PpsbD as a useful promoter to reduce dsRNA accumulation in potato tubers while maintaining the high resistance of the potato leaves to CPB. © 2023 Society of Chemical Industry.

Multifunctional gold nanorods in low-temperature photothermal interactions for combined tumor starvation and RNA interference therapy.

Collateral damage to healthy tissue, uneven heat distribution, inflammatory diseases, and tumor metastasis induction hinder the translation of high-temperature photothermal therapy (PTT) from bench to practical clinical applications. In this report, a multifunctional gold nanorod (GNR)-based nanosystem was designed by attaching siRNA against B7-H3 (B7-H3si), glucose oxidase (GOx), and hyaluronic acid (HA) for efficient low-temperature PTT. Herein, GOx can not only exhaust glucose to induce starvation therapy but also reduce the heat shock protein (HSP), realizing the ablation of tumors without damage to healthy tissues. Evidence shows that B7-H3, a type I transmembrane glycoprotein molecule, plays essential roles in growth, metastasis, and drug resistance. By initiating the downregulation of B7-H3 by siRNA, siRNA-GOx/GNR@HA NPs may promote the effectiveness of treatment. By targeting cluster of differentiation 44 (CD44) and depleting B7-H3 and HSPs sequentially, siRNA-GOx/GNR@HA NPs showed 12.9-fold higher lung distribution than siRNA-GOx/GNR NPs. Furthermore, 50% of A549-bearing mice in the siRNA-GOx/GNR NPs group survived over 50 days. Overall, this low-temperature phototherapeutic nanosystem provides an appropriate strategy for eliminating cancer with high treatment effectiveness and minimal systemic toxicity. STATEMENT OF SIGNIFICANCE: To realize efficient tumor ablation under mild low-temperature (42-45 â„ƒ) and RNA interference simultaneously, here we developed a multifunctional gold nanorod (GNR)-based nanosystem (siRNA-GOx/GNR@HA NPs). This nanoplatform can significantly inhibit tumor cell proliferation and induce cell apoptosis by downregulation of HSP90α, HSP70, B7-H3, p-AKT, and p-ERK and upregulation of cleaved caspase-9 at mild low-temperature due to its superior tumor homing ability and the combined effect of photothermal effect, glucose deprivation-initiated tumor starvation, and B7-H3 gene silence effect. It is believed that this multifunctional low-temperature photothermal nanosystem with efficient and specific anticancer properties, shows a potential application in clinical tumor treatment.

Towards dsRNA-integrated protection of medical Cannabis crops: considering human safety, recent- and developing RNAi methods, and research inroads.

Owing to the expanding industry of medical Cannabis, we discuss recent milestones in RNA interference (RNAi)-based crop protection research and development that are transferable to medical Cannabis cultivation. Recent and prospective increases in pest pressure in both indoor and outdoor Cannabis production systems, and the need for effective nonchemical pest control technologies (particularly crucial in the context of cultivating plants for medical purposes), are discussed. We support the idea that developing RNAi tactics towards protection of medical Cannabis could play a major role in maximizing success in this continuously expanding industry. However, there remain critical knowledge gaps, especially with regard to RNA pesticide biosafety from a human toxicological viewpoint, as a result of the medical context of Cannabis product use. Furthermore, efforts are needed to optimize transformation and micropropagation of Cannabis plants, examine cutting edge RNAi techniques for various Cannabis-pest scenarios, and investigate the combined application of RNAi- and biological control tactics in medical Cannabis cultivation. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Complete protection from Henosepilachna vigintioctopunctata by expressing long double-stranded RNAs in potato plastids.

RNA interference (RNAi) has emerged as a powerful technology for pest management. Previously, we have shown that plastid-mediated RNAi (PM-RNAi) can be utilized to control the Colorado potato beetle, an insect pest in the Chrysomelidae family; however, whether this technology is suitable for controlling pests in the Coccinellidae remained unknown. The coccinellid 28-spotted potato ladybird (Henosepilachna vigintioctopunctata; HV) is a serious pest of solanaceous crops. In this study, we identified three efficient target genes (ß-Actin, SRP54, and SNAP) for RNAi using in vitro double-stranded RNAs (dsRNAs) fed to HV, and found that dsRNAs targeting ß-Actin messenger RNA (dsACT) induced more potent RNAi than those targeting the other two genes. We next generated transplastomic and nuclear transgenic potato (Solanum tuberosum) plants expressing HV dsACT. Long dsACT stably accumulated to up to 0.7% of the total cellular RNA in the transplastomic plants, at least three orders of magnitude higher than in the nuclear transgenic plants. Notably, the transplastomic plants also exhibited a significantly stronger resistance to HV, killing all larvae within 6 d. Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for HV, extending the application range of this technology to Coccinellidae pests.

A Multifunctional Nano-Delivery System Against Rheumatoid Arthritis by Combined Phototherapy, Hypoxia-Activated Chemotherapy, and RNA Interference.

Purpose: Effective therapy for rheumatoid arthritis (RA) keeps a challenge due to the complex pathogenesis of RA. It is not enough to completely inhibit the process of RA with any single therapy method. The purpose of the research is to compensate for the insufficiency of monotherapy using multiple treatment regimens with different mechanisms. Material and Methods: In this study, we developed a new method to synthesize mesoporous silica nanoparticles hybridized with photosensitizer PCPDTBT (HNs). Branched polyethyleneimine-folic acid (PEI-FA) could be coated on the surface of HNs through electrostatic interactions. It simultaneously blocked the hypoxia-activated prodrug tirapazamine loaded into the mesopores and binded with Mcl-1 siRNA (siMcl-1) that interfered with the expression of the anti-apoptotic protein Mcl-1. Released from the co-delivery nanoparticles (PFHNs/TM) Tirapazamine and siMcl-1 upon exposure to acidic conditions of endosomes/lysosomes in activated macrophages. Under NIR irradiation, photothermal therapy and photodynamic therapy derived from PCPDTBT, hypoxia-activated chemotherapy derived from tirapazamine, and RNAi derived from siMcl-1 were used for the combined treatment for RA by killing activated macrophages. PEI-FA-coated PFHNs/TM exhibited activated macrophage-targeting characteristics, thereby enhancing the in vitro and in vivo NIR-induced combined treatment of RA. Results: The prepared PFHNs/TM have high blood compatibility (far below 5% of hemolysis) and ideal in vitro phototherapy effect while controlling the TPZ release and binding siMcl-1. We prove that PEI-FA-coated PFHNs/TM not only protect the bound siRNA but also are selectively uptaked by activated macrophages through FA receptor-ligand-mediated endocytosis, and effectively silence the target anti-apoptotic protein by siMcl-1 transfection. In vivo, PFHNs/TM have also been revealed to be selectively enriched at the inflammatory site of RA, exhibiting NIR-induced anti-RA efficacy. Conclusion: Overall, these FA-functionalized, pH-responsive PFHNs/TM represent a promising platform for the co-delivery of chemical drugs and nucleic acids for the treatment of RA cooperating with NIR-induced phototherapy.

Assessment of a zinc finger protein gene (MPZC3H10) as potential RNAi target for green peach aphid Myzus persicae control.

BACKGROUND: RNA interference (RNAi) has potential application in pest control, and selection of the specific target gene is one of the key steps in RNAi. As an important effector, the zinc finger protein (ZFP) gene has high similarity among aphid species, and may have potential use in an RNAi-based pest control strategy. This study assessed the control efficiency of an RNAi target, MPZC3H10, a CCCH-type ZFP gene, against green peach aphid. RESULTS: ZC3H10 amino acid sequence similarity is more than 97.71% among the five tested aphid species: Myzus persicae, Aphis citricidus, Acyrthosiphon pisum, Diuraphis noxia and Rhopalosiphum maidis. However, no homologous sequence was found in the transcriptome of their ladybeetle predator, Propylaea japonica. Spatial expression patterns revealed that MPZC3H10 showed high expression in the muscle and fat body of M. persicae. The RNAi bioassay revealed that silencing of MPZC3H10 resulted in high mortality (53.33%) in M. persicae. By contrast, there were no observed negative effects on the growth and development of P. japonica when fed on aphids treated with double-stranded RNA (dsRNA) or injected with a "high dose" of dsRNA. CONCLUSION: Targeting MPZC3H10 showed promising efficiency for green peach aphid control via artificially designed dsRNA, and was safe for the predatory ladybeetle. © 2022 Society of Chemical Industry.

RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

Potato (Solanum tuberosum L.) non-specific lipid transfer protein StLTP6 promotes viral infection by inhibiting virus-induced RNA silencing.

MAIN CONCLUSION: For the first time it is reported that members of the nsLTP protein family could promote viral infection by inhibiting virus-induced RNA silencing. Non-specific lipid transfer proteins (nsLTPs) are a class of soluble proteins with low relative molecular weight and widely present in higher plants. The role of nsLTPs in biotic and abiotic stresses has been studied, but no report has shown that nsLTPs play a role in the process of viral infection. We report the function and mechanism of the classical nsLTP protein StLTP6 in viral infection. We found that StLTP6 expression was remarkably upregulated in potato infected with potato virus Y and potato virus S. The infection efficiency and virus content of StLTP6-overexpressed potato and Nicotiana benthamiana were remarkable increased. Further study found that the overexpression of StLTP6 inhibited the expression of multiple genes in the RNA silencing pathway, thereby inhibiting virus-induced RNA silencing. This result indicated that StLTP6 expression was induced during viral infection to inhibit the resistance of virus-induced RNA silencing and promote viral infection. In summary, we reported the role of StLTP6 in viral infection, broadening the biological function range of the nsLTP family and providing valuable information for the study of viral infection mechanism.

Impact of Exogenous Application of Potato Virus Y-Specific dsRNA on RNA Interference, Pattern-Triggered Immunity and Poly(ADP-ribose) Metabolism.

In this work we developed and exploited a spray-induced gene silencing (SIGS)-based approach to deliver double-stranded RNA (dsRNA), which was found to protect potato against potato virus Y (PVY) infection. Given that dsRNA can act as a defence-inducing signal that can trigger sequence-specific RNA interference (RNAi) and non-specific pattern-triggered immunity (PTI), we suspected that these two pathways may be invoked via exogeneous application of dsRNA, which may account for the alterations in PVY susceptibility in dsRNA-treated potato plants. Therefore, we tested the impact of exogenously applied PVY-derived dsRNA on both these layers of defence (RNAi and PTI) and explored its effect on accumulation of a homologous virus (PVY) and an unrelated virus (potato virus X, PVX). Here, we show that application of PVY dsRNA in potato plants induced accumulation of both small interfering RNAs (siRNAs), a hallmark of RNAi, and some PTI-related gene transcripts such as WRKY29 (WRKY transcription factor 29; molecular marker of PTI), RbohD (respiratory burst oxidase homolog D), EDS5 (enhanced disease susceptibility 5), SERK3 (somatic embryogenesis receptor kinase 3) encoding brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and PR-1b (pathogenesis-related gene 1b). With respect to virus infections, PVY dsRNA suppressed only PVY replication but did not exhibit any effect on PVX infection in spite of the induction of PTI-like effects in the presence of PVX. Given that RNAi-mediated antiviral immunity acts as the major virus resistance mechanism in plants, it can be suggested that dsRNA-based PTI alone may not be strong enough to suppress virus infection. In addition to RNAi- and PTI-inducing activities, we also showed that PVY-specific dsRNA is able to upregulate production of a key enzyme involved in poly(ADP-ribose) metabolism, namely poly(ADP-ribose) glycohydrolase (PARG), which is regarded as a positive regulator of biotic stress responses. These findings offer insights for future development of innovative approaches which could integrate dsRNA-induced RNAi, PTI and modulation of poly(ADP-ribose) metabolism in a co-ordinated manner, to ensure a high level of crop protection.