Plant-mediated RNA Interference Research Articles

Efficacy of arginine kinase as a promising RNAi target in Aphis gossypii genome as revealed through aphid bioassay on field-grown transgenic cotton plants

The cotton aphid Aphis gossypii is a major agricultural pest of cotton that causes substantial damage to the crop not only by sucking sap but also through virus transmission. Globally adopted traditional and contemporary approaches to control aphid infestation have certain limitations and are hazardous to human health. RNA interference (RNAi) technology has unfolded its potential as an effective crop protection strategy against various pests. In this study, we adopted plant-mediated RNAi strategy to enhance aphid resistance in cotton by targeting arginine kinase (AK), which is a crucial enzyme responsible for energy homeostasis in insects. We selected a 312bp dsRNA fragment containing eight siRNAs and showing optimum GC content, Hb index, and stable secondary structure based on computational prediction studies. The binary construct expressing dsRNA was used to transform local cotton variety MNH886 and four transgenic lines were obtained in the T1 generation. Out of the four T1 transgenic cotton lines, dsA-7 exhibited the highest aphid mortality (73.3%), whereas, dsA-1, dsA-3 and dsA-6 revealed 60%, 61%, and 66.6% aphid mortality, respectively, in comparison to 13.3% mortality in the mock control cotton line. Moreover, significant knockdown in mRNA expression of AK was observed in aphids fed dsA-7 which was 79%. In comparison, 54%, 47%, and 45% downregulation was recorded in aphids which fed on dsA-6, dsA-3, and dsA-1 transgenic cotton lines, respectively. These results revealed that plant-mediated downregulation of aphid RNA induced significant RNA interference in A. gossypii which resulted in considerable aphid mortality and led to plant protection against aphids.

Harnessing plant-mediated RNAi for effective management of Phthorimaea absoluta by targeting AChE1 and SEC23 genes

Tomato production on a global scale is under persistent pressure due to the devastating impact of Phthorimaea absoluta Meyrick (Lepidoptera: Gelechiidae), the South American tomato leaf miner. To combat this devastating pest, we explored the potential of plant-mediated RNA interference (RNAi) as a novel strategy for its management. Using transgenic techniques, we developed RNAi constructs (p35S::dsAChE1, p35S::dsSEC23) targeting crucial genes, AChE1 and SEC23, in P. absoluta. These genes play pivotal roles in insect physiology and development. The transformation of tomato cultivar Rio Grande was carried out with these RNAi constructs using Agrobacterium tumefaciens. The results demonstrated a significant reduction in transcript levels of both AChE1 and SEC23 in P. absoluta. Silencing AChE1 resulted in substantial mortality rates, reduced larval weight gain, and deformities, highlighting its pivotal role in insect survival. SEC23 gene silencing also induced mortality and influenced insect physiology. Furthermore, we explored the susceptibility of AChE1 to organophosphate insecticides, revealing its relevance in insecticide susceptibility. These findings support the potential of AChE1 and SEC23 as valuable targets for RNAi-based control of P. absoluta for the first time, providing multifaceted insights into insect physiology and insecticide susceptibility, thereby offering valuable insights for the development of effective strategies to mitigate the impact of this destructive pest.

Knockdown of orthotospovirus-derived silencing suppressor gene by plant-mediated RNAi approach induces viral resistance in tomato

RNA interference (RNAi) shows significant effectiveness in conferring resistance to viral infections in various plants. The current study was conducted to develop transgenic tomato (Solanum lycopersicum cv. Rio Grande) lines expressing a 320 bp conserved inverted region of the integral 35S promoter region. The molecular cloning was done for the construction of expression vectors in a hairpin structure by the Tomato spotted wilt orthotospovirus (TSWV) species Orthotospovirus tomatomaculae, silencing suppressor (NSs) gene. It contains both sense and antisense orientation in a binary vector pFGC5941. Short hairpin RNA (shRNA) structures were employed to reduce the possibility of inducing endogenous non-specific antiviral responses, which usually target longer double-stranded RNAs (dsRNAs). To confirm the transgenic plants, molecular analysis was performed using TSWV gene-specific primers (TSWV-NSs). Based on the EHA105 Agrobacterium strain and the pFGC5941 vector carrying NSs and nptII genes in the T-DNA region, the results validate the insertion of kanamycin resistance into the regenerated transgenic plants. The virus source harboring TSWV was used to inoculate putative transgenic plants for evaluating the confirmation of successful transformation strategy for inducing virus resistance. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed by using qRT-NSs primer pairs to determine the relative gene expression of all constructs (TSWV-NSs both sense and antisense orientation) after inoculations. In comparison to the control groups, which contained wild type control (non-transgenic tomato plant), empty pFGC5941 vector and the pFGC5941+sense construct, the expression of the NSs gene was noticeably reduced in the full clone (pFGC5941+sense + antisense) construct. For the validation of RNAi effectiveness, Nicotiana tabacum plants were also inoculated with viral constructs. Specifically, they displayed severe symptoms of TSWV in plants containing the pFGC5941+sense, pFGC5941 empty vector, and in wild-type tomato plants that are correlated with bioassay results. The lack of TSWV symptoms validated the hypothesis after careful observation of the expression of NSs genes in terms of pathogenicity. This research demonstrates the RNA interference effectiveness by targeting NSs gene as a reliable technique for achieving long lasting antiviral protection in tomatoes. The findings have practical relevance and the potential to be a tool for limiting tomato crop losses caused by TSWV worldwide.

Host-induced silencing of MpPar6 confers Myzus persicae resistance in transgenic rape plants

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for insect control. The green peach aphid, Myzus persicae, feeds on over 400 species of host plants. Brassica napus (rape) is the second most important oilseed crop worldwide. Myzus persicae is highly reproductive and causes severe damage to the rape plants due to its quite flexible life cycle. In this study, we tested the RNAi effects of transgenic rape plants on M. persicae. By in vitro feeding M. persicae with artificial diets containing double-stranded RNAs (dsRNAs) targeting seven aphid genes, we identified a new gene encoding the partitioning-defective protein 6 (Par6) as the most potent RNAi target. Tissue- and stage-expression analysis of Par6 suggested this gene is highly expressed in the embryo and adult stage of M. persicae. We next generated transgenic rape plants expressing dsPar6 by Agrobacterium-mediated transformation and obtained nine independent transgenic lines. Compared to wild-type control plants, transgenic rape lines expressing dsPar6 showed strong resistance to M. persicae. Feeding assays revealed that feeding transgenic rape plants to M. persicae significantly decreased MpPar6 expression and survival rate and impaired fecundity. Furthermore, we showed that the resistance levels to M. persicae are positively correlated with dsPar6 expression levels in transgenic rape plants. Our study demonstrates that transgenic rape plants expressing dsPar6 are efficiently protected from M. persicae. Interfering with the genes involved in embryo development could be the effective RNAi targets for controlling aphids and potentially other insect pests.

Control of white mold (Sclerotinia sclerotiorum) through plant-mediated RNA interference

The causative agent of white mold, Sclerotinia sclerotiorum, is capable of infecting over 600 plant species and is responsible for significant crop losses across the globe. Control is currently dependent on broad-spectrum chemical agents that can negatively impact the agroecological environment, presenting a need to develop alternative control measures. In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing hairpin (hp)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 compared to wild-type Col-0. To better understand how HIGS influences host–pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 directly at the site of S. sclerotiorum infection. RNA sequencing data reveals enrichment of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) pathway, as well as transcription factors predicted to regulate plant immunity. Using RT-qPCR, we identified predicted interacting partners of ABHYDROLASE-3 in the polyamine synthesis pathway of S. sclerotiorum that demonstrate co-reduction with ABHYDROLASE-3 transcript levels during infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provide insight into the role of ABHYDROLASE-3 in the A. thaliana–S. sclerotiorum pathosystem.

Horizontally transferred genes as RNA interference targets for aphid and whitefly control.

RNA interference (RNAi)-based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi or plants, are attractive targets for RNAi-mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco-adapted isolate of Myzus persicae (green peach aphid) and bioinformatically identified 30 HTGs. We then used plant-mediated virus-induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal-origin HTGs did not affect aphid survivorship but decreased aphid reproduction. Importantly, although there was uptake of plant-expressed RNA by Coccinella septempunctata (seven-spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid-targeted VIGS constructs. To demonstrate that this approach is more broadly applicable, we also targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant-mediated RNA interference.

Silencing an aphid-specific gene SmDSR33 for aphid control through plant-mediated RNAi in wheat.

Grain aphid (Sitobion miscanthi) is one of the most dominant and devastating insect pests in wheat, which causes substantial losses to wheat production each year. Engineering transgenic plants expressing double strand RNA (dsRNA) targeting an insect-specific gene has been demonstrated to provide an alternative environmentally friendly strategy for aphid management through plant-mediated RNA interference (RNAi). Here we identified and characterized a novel potential RNAi target gene (SmDSR33) which was a gene encoding a putative salivary protein. We then generated stable transgenic wheat lines expressing dsRNA for targeted silencing of SmDSR33 in grain aphids through plant-mediated RNAi. After feeding on transgenic wheat plants expressing SmDSR33-dsRNA, the attenuated expression levels of SmDSR33 in aphids were observed when compared to aphids feeding on wild-type plants. The decreased SmDSR33 expression levels thus resulted in significantly reduced fecundity and survival, and decreased reproduction of aphids. We also observed altered aphid feeding behaviors such as longer duration of intercellular stylet pathway and shorter duration of passive ingestion in electroneurography assays. Furthermore, both the surviving aphids and their offspring exhibited decreased survival rates and fecundity, indicating that the silencing effect could be persistent and transgenerational in grain aphids. The results demonstrated that SmDSR33 can be selected as an effective RNAi target for wheat aphid control. Silencing of an essential salivary protein gene involved in ingestion through plant-mediated RNAi could be exploited as an effective strategy for aphid control in wheat.

Effects of transgenic cotton lines expressing dsAgCYP6CY3-P1 on the growth and detoxification ability of Aphis gossypii glover.

The pest Aphis gossypii Glover globally causes considerable economic losses on various crops by its feeding damage and disease transmission. Transgenic plants that produce double-stranded RNA (dsRNA) targeted to insect genes are being developed as a pest control strategy. In this study, we evaluated the effects of transgenic cotton-mediated RNA interference (RNAi) on the growth and detoxification ability of A. gossypii after the transgenic cotton lines expressing dsAgCYP6CY3-P1 (the TG cotton lines) were obtained on the basis of exploring the functions of CYP6CY3 in our previous research. The developmental time of third- and fourth-instar nymphs which fed on the TG cotton lines were significantly prolonged. Life table parameters showed that the fitness of cotton aphids from the TG cotton lines decreased. Additionally, the relative expression level of CYP6CY3 in cotton aphids which fed on the TG cotton lines was significantly reduced by 47.3 % at 48 h compared with that from the nontransgenic cotton (the NT cotton). Bioassay showed that silencing of CYP6CY3 increased mortality of the nymphs to imidacloprid by 28.49 % (at 24 h) and to acetamiprid by 73.77 % (at 48 h), respectively. These results indicated that the TG cotton lines delayed the growth and development of A. gossypii, but also decreased population density and increased its sensitivity to imidacloprid and acetamiprid, respectively. The results provide further support for the development and application of plant-mediated RNAi. © 2022 Society of Chemical Industry.

A highly accumulated secretory protein from cotton bollworm interacts with basic helix-loop-helix transcription factors to dampen plant defense.

Caterpillar oral secretion (OS) contains active molecules that modulate plant defense signaling. We isolated an effector-like protein (Highly Accumulated Secretory Protein 1, HAS1) from cotton bollworm (Helicoverpa armigera) that is the most highly accumulated secretory protein of the nondigestive components in OS and belongs to venom R-like protein. Elimination of HAS1 by plant-mediated RNA interference reduced the suppression of OS on the defense response in plants. Plants expressing HAS1 are more susceptible to insect herbivory accompanied by the reduced expressions of multiple defense genes. HAS1 binds to the basic helix-loop-helix (bHLH) transcription factors, including GoPGF involved in pigmented gland formation and defense compounds biosynthesis in cotton and MYC3/MYC4 the main regulators in jasmonate (JA) signaling in Arabidopsis. The binding activity is required for HAS1 to inhibit the activation of bHLHs on plant defense gene expressions. Together with our previous study that another venom R-like protein HARP1 in cotton bollworm OS blocks JA signaling by interacting with JASMONATE-ZIM-domain repressors, we conclude that the venom R-like proteins in OS interfere with plant defense in a dual suppression manner. Considering the venom proteins in parasitic wasp assault the immune system of its host animal, our investigation reveals their conserved function in carnivorous and herbivorous insects.

FAR knockout significantly inhibits Chilo suppressalis survival and transgene expression of double-stranded FAR in rice exhibits strong pest resistance.

Chilo suppressalis is one of the most prevalent and damaging rice pests, causing significant economic losses each year. Chemical control is currently the primary method of controlling C. suppressalis. However, the indiscriminate use of chemical insecticides increases pest resistance, pollutes the environment and poses a significant health threat to humans and livestock, highlighting the need to find safer, more pest-specific and more effective alternatives to pest control. Plant-mediated RNA interference (RNAi) is a promising agricultural pest control method that is highly pest-specific and has less of an impact on the environment. Using multi-sgRNAs/Cas9 technology to delete Fatty acyl-CoA reductase (FAR) of C. suppressalis in the G0 generation, we show that downregulating FAR transcription may significantly increase the mortality rate and darken the epidermis of C. suppressalis compared with the control. Subsequently, we developed dsFAR transgenic rice lines using Agrobacterium-mediated genetic transformation and then screened three strains expressing dsFAR at high levels using transcriptional level analysis. Using transgenic rice stems, a laboratory feeding bioassay indicated that at least one line (L#10) displayed a particularly high level of insect resistance, with an insect mortality rate of more than 80%. In the field trials, dsFAR transgenic rice displayed high levels of resistance to C. suppressalis damage. Collectively, these results suggest the potential of a new environment-friendly, species-specific strategy for rice pest management.

Application progress of plant-mediated RNAi in pest control.

RNA interference (RNAi)-based biopesticides are novel biologic products, developed using RNAi principles. They are engineered to target genes of agricultural diseases, insects, and weeds, interfering with their target gene expression so as to hinder their growth and alleviate their damaging effects on crops. RNAi-based biopesticides are broadly classified into resistant plant-based plant-incorporated protectants (PIPs) and non-plant-incorporated protectants. PIP RNAi-based biopesticides are novel biopesticides that combine the advantages of RNAi and resistant transgenic crops. Such RNAi-based biopesticides are developed through nuclear or plastid transformation to breed resistant plants, i.e., dsRNA-expressing transgenic plants. The dsRNA of target genes is expressed in the plant cell, with pest and disease control being achieved through plant-target organism interactions. Here, we review the action mechanism and strategies of RNAi for pest management, the development of RNAi-based transgenic plant, and the current status and advantages of deploying these products for pest control, as well as the future research directions and problems in production and commercialization. Overall, this study aims to elucidate the current development status of RNAi-based biopesticides and provide guidelines for future research.

Silencing a Myzus persicae Macrophage Inhibitory Factor by Plant-Mediated RNAi Induces Enhanced Aphid Mortality Coupled with Boosted RNAi Efficacy in Transgenic Potato Lines.

Myzus persicae causes considerable losses to crops as a major pest. The damage is direct by feeding and also partly indirect because it vectors plant viruses. The currently available control strategies rely on unsafe and nonecofriendly chemical pesticide applications. Plant-mediated RNA interference (RNAi) has emerged as a powerful tool in crop protection from insect pests. Aphid salivary proteins are essential for phloem feeding and act as mediators of the complex interactions between aphids and their host plants. We documented the efficacy of dsRNA directed against macrophage inhibitory factor (MIF1) of M. persicae to induce aphid mortality and gene silencing through the generation of transgenic potato lines. A binary construct harbouring dsMIF1 driven by the CaMV35S promoter was introduced into the local potato variety 'AGB-white' by Agrobacterium-mediated transformation. PCR and Southern blotting validated the transgene presence and genomic integration in seven transgenic potato lines. An in vitro detached leaf assay revealed a significantly high aphid mortality of 65% in the transgenic potato line sDW-2, while the aphid mortality was 77% in the sDW-2 transgenic line during the in planta bioassay in comparison with 19% aphid mortality in the control nontransgenic potato line. A significantly high silencing effect was observed in the mRNA expression of MIF1, which was reduced to 21% in aphids fed on the transgenic potato line sDW-2. However, variable knockdown effects were found among six other transgenic potato lines, ranging from 30 to 62%. The study concluded that plant-mediated silencing of aphid RNA induces significant RNAi in M. persicae, along with enhanced aphid mortality.

Resistance to RNA interference by plant‐derived double‐stranded RNAs but not plant‐derived short interfering RNAs in Helicoverpa armigera

Plant-mediated RNA interference (RNAi) has emerged as a promising technology for pest control through expression of double-stranded RNAs (dsRNAs) targeted against essential insect genes. However, little is known about the underlying molecular mechanisms and whether long dsRNA or short interfering RNAs (siRNAs) are the effective triggers of the RNAi response. Here we generated transplastomic and nuclear transgenic tobacco plants expressing dsRNA against the Helicoverpa armigera ATPaseH gene. We showed that expression of long dsRNA of HaATPaseH was at least three orders of magnitude higher in transplastomic plants than in transgenic plants. HaATPaseH-derived siRNAs are absent from transplastomic plants, while they are abundant in transgenic plants. Feeding transgenic plants to H. armigera larvae reduced gene expression of HaATPaseH and delayed growth. Surprisingly, no effect of transplastomic plants on insect growth was observed, despite efficient dsRNA expression in plastids. Furthermore, we found that dsRNA ingested by H. armigera feeding on transplastomic plants was rapidly degraded in the intestinal fluid. In contrast, siRNAs are relatively stable in the digestive system. These results suggest that plant-derived siRNAs may be more effective triggers of RNAi in Lepidoptera than dsRNAs, which will aid the optimization of the strategies for plant-mediated RNAi to pest control.

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species.

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects.

Inhibition of Rhopalosiphum maidis (Corn Leaf Aphid) Growth on Maize by Virus-Induced Gene Silencing with Sugarcane Mosaic Virus.

The corn leaf aphid (Rhopalosiphum maidis), a damaging pest of maize (Zea mays), is not controlled by the insecticidal proteins in commercially available transgenic crop varieties. One promising approach is to reduce aphid growth and fecundity by targeting the expression of essential genes using plant-mediated RNA interference (RNAi). Here we describe a method whereby Sugarcane Mosaic Virus (SCMV), a positive-strand RNA virus in the Potyviridae family, is used for virus-induced gene silencing (VIGS) of gene expression in R. maidis. A segment of the R. maidis target gene is cloned into SCMV, maize plants are infected with the transgenic virus, aphids are placed on the virus-infected plants and, after a few days of feeding, decreases in target gene expression and aphid reproduction are assessed. This VIGS method can be used for rapid screening of suitable RNAi targets for aphid pest control, as well as to study the in vivo function of specific aphid genes.

Rapid Screening of Myzus persicae (Green Peach Aphid) RNAi Targets Using Tobacco Rattle Virus.

Plant-mediated RNA interference (RNAi) can be used to reduce the growth of insect pests, including Myzus persicae (green peach aphid), a prolific pest of numerous dicot crop species. In one approach, viruses that have been engineered to carry an aphid gene fragment are used to infect plants and thereby silence target gene expression in the aphids feeding on these plants, a process called virus-induced gene silencing, or VIGS. Tobacco Rattle Virus (TRV) in the model plant, Nicotiana benthamiana, was the first of many VIGS systems that have been developed for different plant species. In this chapter, we describe a method for silencing M. persicae gene expression using an established TRV-VIGS vector that infects and spreads in N. benthamiana. The two parts of the TRV genome, RNA1 and RNA2, have been cloned into Agrobacterium T-DNA vectors for initiation of plant infections. The RNA2 construct is modified with a Gateway-compatible cloning site to allow insertion of aphid genes. When feeding on TRV-infected N. benthamiana plants, aphids ingest dsRNAs that silence specific target genes. TRV-VIGS of aphid genes allows rapid identification of essential gene targets that canbe used for the control of M. persicae by this and other RNAi methods.

Transplastomic Tomato Plants Expressing Insect-Specific Double-Stranded RNAs: A Protocol Based on Biolistic Transformation.

Expressing insecticidal double-stranded RNA (dsRNA) molecules in plant plastids is a novel approach for in planta production of dsRNA that has enormous potential for developing improved plant-mediated RNA interference (RNAi) strategies for insect pest control. In this chapter, we describe the design of a transformation vector containing an expression cassette which can be used to stably transform plastids of tomato plants for production and accumulation of dsRNA . Such dsRNA can trigger the mechanisms of RNAi in pest insects and selectively suppress the expression of target genes, resulting in lethality. We also describe a protocol for detection of full-length dsRNA molecules in plastids using an RT-PCR-based method.

Plant-Mediated RNA Interference Expressing dsRNA in Cytoplasm for RNAi-Based Pest Control.

Currently, RNAi-based techniques have exhibited considerable potential for pest control, and considerable progress has been made in identifying a large number of potential target genes in the laboratory. However, applying potential target genes effectively in the field to control pests requires further research. At present, transgenic crops expressing dsRNA are widely recognized as the most practical and economical means of applying RNAi-based techniques using laboratory-identified target genes to control pests in the field. Among the three plant-mediated RNAi techniques, plant-mediated RNA interference expressing dsRNA in cytoplasm is most often considered to be an economical and practical RNAi method for pest control, both in the laboratory and inthe field. In this chapter, I describe the procedure for expressing dsRNA in plant cytoplasm for RNAi-based pest control.

Transgenic double-stranded RNA rice, a potential strategy for controlling striped stem borer (Chilo suppressalis).

Although the striped stem borer (SSB, Chilo suppressalis Walker) is a devastating pest of rice that causes significant economic losses, management options are currently limited. Plant-mediated RNA interference (RNAi) is an emerging crop protection technique in which transgenic plants are modified to express insect-specific double-stranded RNAs (dsRNAs) that trigger RNAi silencing in target pests. In this study, an RNAi-based screen of 35 candidate SSB genes identified a small heat shock protein gene (CssHsp) as a potential plant-based RNAi target. To assess its utility in planta, a total of 39 transgenic rice plants were generated, with 11 independent transformants found to contain a single copy of the dsCssHsp expression cassette. In life-time feeding bioassays, three transgenic lines (DS10, DS35, DS36) were found to have significant negative impacts on SSB populations. After feeding for 8 days, mortality in the three transgenic lines exceeded 60%. By pupation, mortality further increased to 90% and few SSB survived to eclosion. Gene expression analyses confirmed that CssHsp transcript levels were significantly reduced after feeding on the transgenic dsCssHsp rice. These results demonstrate the potential for developing a plant-mediated RNAi strategy targeting CssHsp as a more biorational field-based approach for SSB control. © 2021 Society of Chemical Industry.

Detoxification of plant defensive glucosinolates by an herbivorous caterpillar is beneficial to its endoparasitic wasp.

Plant chemical defences impact not only herbivores, but also organisms in higher trophic levels that prey on or parasitize herbivores. While herbivorous insects can often detoxify plant chemicals ingested from suitable host plants, how such detoxification affects endoparasitoids that use these herbivores as hosts is largely unknown. Here, we used transformed plants to experimentally manipulate the major detoxification reaction used by Plutella xylostella (diamondback moth) to deactivate the glucosinolate defences of its Brassicaceae host plants. We then assessed the developmental, metabolic, immune, and reproductive consequences of this genetic manipulation on the herbivore as well as its hymenopteran endoparasitoid Diadegma semiclausum. Inhibition of P.xylostella glucosinolate metabolism by plant-mediated RNA interference increased the accumulation of the principal glucosinolate activation products, the toxic isothiocyanates, in the herbivore, with negative effects on its growth. Although the endoparasitoid manipulated the excretion of toxins by its insect host to its own advantage, the inhibition of herbivore glucosinolate detoxification slowed endoparasitoid development, impaired its reproduction, and suppressed the expression of genes of a parasitoid-symbiotic polydnavirus that aids parasitism. Therefore, the detoxification of plant glucosinolates by an herbivore lowers its toxicity as a host and benefits the parasitoid D.semiclausum at multiple levels.