Market Barriers to RNAi Adoption in Agriculture: Evidence From a Multi‐Country Discrete Choice Experiment Amongst European Consumers

RNAinterference (RNAi) technology is considered an alternative tool to develop more environmentally friendly broad-spectrumpesticides in agriculture. In this approach, sequence-specific knockdown of gene targets in pests and pathogensusing double-strandedRNA(dsRNA) is utilized. Two different dsRNAapplicationmethods, host induced gene silencing (HIGS) and spray induced gene silencing (SIGS) are being followed. HIGS involves developing transgenic plants that produce the intended dsRNA which will be delivered into the pests when they feed or grow on the transgenic plants, while in SIGS the dsRNAs applied topically on the plants will be taken up by the target organisms. Once the dsRNA is in the target organism, the host RNAi cellular machinery will be used to silence the target genes. SIGS has been applied now against many pests and diseases in different crops and has given promising results. With the development of tools that facilitate economic production of large scale dsRNA and improve the stability and longevity of the sprayed dsRNAs on the plant surface, SIGS is a promising technology that could be adopted across crops and against different pests and pathogens. In this research update, we provide a summary of the recent developments in the area of SIGS with an emphasis on the examples of fungal pathogen control.

Thysanoptera, commonly known as thrips, are agricultural pests capable of causing significant crop damage and serving as vectors for plant viruses. RNA interference (RNAi) has been widely studied as a potential approach for pest management due to its species-specific gene silencing mechanism. This review summarizes current research on RNAi mechanisms in Thysanoptera, including dsRNA uptake, intracellular transport, and gene silencing pathways. Various RNAi-based pest control strategies, such as host-induced gene silencing (HIGS), spray-induced gene silencing (SIGS), and microbe-induced gene silencing (MIGS), are discussed, along with the challenges associated with their implementation. In addition, recent studies on nanoparticle-mediated dsRNA delivery are reviewed, with an emphasis on improving RNAi efficiency and stability under field conditions. Further, RNAi applications in addressing insecticide resistance and reducing viral transmission in thrips are examined. While RNAi presents a potential alternative to traditional chemical control methods, uncertainties remain regarding its systemic transport, environmental persistence, and regulatory assessment. By summarizing recent findings, this review aims to provide an updated perspective on the feasibility and limitations of RNAi technology in Thysanoptera pest management.

Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.

Plant pathogenic fungi and oomycetes cause severe losses of crop yield worldwide. Fungicides are widely applied to manage plant diseases caused by pathogenic fungi, but fungicide-resistant fungal populations have been increasingly reported. Recent techniques using RNA interference (RNAi), which define the ability of double-stranded RNA (dsRNA) to inhibit the expression of homologous gene(s), have been suggested for crop protection in an environmental-friendly way. These techniques, so-called host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), are the innovative strategies to control plant diseases. The HIGS involves host expression of dsRNA targeting genes in interacting plant pathogens and the SIGS involves inhibition of plant pathogens through a direct spray of dsRNA targeting pathogen genes on plant tissues. In this review, we present recent studies of the HIGS and SIGS to protect plant diseases caused by fungal and oomycete pathogens.

Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.

Chapter 6 - RNA interference as a promising strategy for plant disease management

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.

Efforts to develop more environmentally friendly alternatives to traditional broad-spectrum pesticides in agriculture have recently turned to RNA interference (RNAi) technology. With the built-in, sequence-specific knockdown of gene targets following delivery of double-stranded RNA (dsRNA), RNAi offers the promise of controlling pests and pathogens without adversely affecting non-target species. Significant advances in the efficacy of this technology have been observed in a wide range of species, including many insect pests and fungal pathogens. Two different dsRNA application methods are being developed. First, host induced gene silencing (HIGS) harnesses dsRNA production through the thoughtful and precise engineering of transgenic plants and second, spray induced gene silencing (SIGS) that uses surface applications of a topically applied dsRNA molecule. Regardless of the dsRNA delivery method, one aspect that is critical to the success of RNAi is the ability of the target organism to internalize the dsRNA and take advantage of the host RNAi cellular machinery. The efficiency of dsRNA uptake mechanisms varies across species, and in some uptake is negligible, rendering them effectively resistant to this new generation of control technologies. If RNAi-based methods of control are to be used widely, it is critically important to understand the mechanisms underpinning dsRNA uptake. Understanding dsRNA uptake mechanisms will also provide insight into the design and formulation of dsRNAs for improved delivery and provide clues into the development of potential host resistance to these technologies.

RNA interference (RNAi) is a biotechnological tool used for gene silencing in plants, with both endogenous and exogenous applications. Endogenous approaches, such as host-induced gene silencing (HIGS), involve genetically modified (GM) plants, while exogenous methods include spray-induced gene silencing (SIGS). The RNAi mechanism hinges on the introduction of double-stranded RNA (dsRNA), which is processed into short interfering RNAs (siRNAs) that degrade specific messenger RNAs (mRNAs). However, unintended effects on non-target organisms and GM plants are a concern due to sequence homologies or siRNA-induced epigenetic changes. Regulatory bodies such as the EPA and EFSA emphasize the need for comprehensive risk assessments. Detecting unintended effects is complex, often relying on bioinformatic tools and untargeted analyses like transcriptomics and metabolomics, though these methods require extensive genomic data. This review aims to classify mechanisms of RNAi effects induced by short interfering RNA from different sources in plants and to identify technologies that can be used to detect these effects. In addition, practical case studies are summarized and discussed in which previously unintended RNAi effects in genetically modified plants have been investigated. Current literature is limited but suggests RNAi is relatively specific, with few unintended effects observed in GM crops. However, further studies are needed to fully understand and mitigate potential risks, particularly those related to transcriptional gene silencing (TGS) mechanisms, which are less predictable than post-transcriptional gene silencing (PTGS). Particularly the application of untargeted approaches such as small RNA sequencing and transcriptomics is recommended for thorough and comprehensive risk assessments.

Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production.

RNA based gene silencing modalities to control insect and fungal plant pests – Challenges and future prospects

Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley (Hordeum vulgare) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum. Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.

Since the beginning of agriculture, plant virus diseases have been a strong challenge for farming. Following its discovery at the very beginning of the 1990s, the RNA interference (RNAi) mechanism has been widely studied and exploited as an integrative tool to obtain resistance to viruses in several plant species, with high target-sequence specificity. In this chapter, we describe and review the major aspects of host-induced gene silencing (HIGS), as one of the possible plant defence methods, using genetic engineering techniques. In particular, we focus our attention on the use of RNAi-based gene constructs to introduce stable resistance in host plants against viral diseases, by triggering post-transcriptional gene silencing (PTGS). Recently, spray-induced gene silencing (SIGS), consisting of the topical application of small RNA molecules to plants, has been explored as an alternative tool to the stable integration of RNAi-based gene constructs in plants. SIGS has great and innovative potential for crop defence against different plant pathogens and pests and is expected to raise less public and political concern, as it does not alter the genetic structure of the plant.

Since the beginning of agriculture, plant virus diseases have been a strong challenge for farming. Following its discovery at the very beginning of the 1990s, the RNA interference (RNAi) mechanism has been widely studied and exploited as an integrative tool to obtain resistance to viruses in several plant species, with high target-sequence specificity. In this chapter, we describe and review the major aspects of host-induced gene silencing (HIGS), as one of the possible plant defence methods, using genetic engineering techniques. In particular, we focus our attention on the use of RNAi-based gene constructs to introduce stable resistance in host plants against viral diseases, by triggering post-transcriptional gene silencing (PTGS). Recently, spray-induced gene silencing (SIGS), consisting of the topical application of small RNA molecules to plants, has been explored as an alternative tool to the stable integration of RNAi-based gene constructs in plants. SIGS has great and innovative potential for crop defence against different plant pathogens and pests and is expected to raise less public and political concern, as it does not alter the genetic structure of the plant.

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