Fungal small RNA: unveiling the breakthroughs and promising applications.

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.

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 diseases cause significant decreases in yield and quality of crops and consequently pose a very substantial threat to food security. In the continuous search for environmentally friendly crop protection, exploitation of RNA interferance machinery is showing promising results. It is well established that small RNAs (sRNAs) including microRNA (miRNA) and small interfering RNA (siRNA) are involved in the regulation of gene expression via both transcriptional and post-transcriptional RNA silencing. sRNAs from host plants can enter into pathogen cells during invasion and silence pathogen genes. This process has been exploited through Host-Induced Gene Silencing (HIGS), in which plant transgenes that produce sRNAs are engineered to silence pest and pathogen genes. Similarly, exogenously applied sRNAs can enter pest and pathogen cells, either directly or via the hosts, and silence target genes. This process has been exploited in Spray-Induced Gene Silencing (SIGS). Here, we focus on the role of sRNAs and review how they have recently been used against various plant pathogens through HIGS or SIGS-based methods and discuss advantages and drawbacks of these approaches.

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.

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.

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

Small RNAs (sRNAs) are short, non-coding RNA molecules that play a crucial role in regulating gene expression. They include microRNAs (miRNAs) and small interfering RNAs (siRNAs) and are involved in controlling plant immunity against pathogens and pests. Recent research has revealed that sRNAs can move between different species and silence genes, a process called cross-kingdom RNA interference (RNAi). This discovery has led to the development of host-induced gene silencing (HIGS), where transgenic plants are engineered to produce double-stranded RNA (dsRNA) that targets specific pathogen genes. This RNA-based approach offers a promising alternative to agrochemicals and transgenic crops with potential health and environmental risks. By applying dsRNAs or sRNAs directly onto host plants or post-harvest products, a technique known as spray-induced gene silencing (SIGS), effective disease management can be achieved without the need for transgenic methods. This chapter explores recent advancements in using cross-kingdom RNAi to manage plant diseases

Host-induced gene silencing (HIGS) is a common method for engineering plant protection against pathogens, although success requires double-stranded RNA (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: (i) S. musiva’s capacity for uptake of environmental dsRNA; (ii) effects of in vitro silencing of CYP51 and DCL on fungal growth and target transcript abundance; and (iii) persistence of dsRNA in culture. The 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 whether HIGS can be an effective technology for limiting stem canker. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

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.

Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) via double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack. Yet, due to biosafety concerns regarding transgenics, country-specific GMO legislation has limited the practical application of desirable attributes in plants. The topical application of dsRNA/siRNA targeting essential fungal gene(s) through spray-induced gene silencing (SIGS) on host plants has opened up a transgene-free avenue for crop protection. However, several factors influence the outcome of RNAi, including but not limited to RNAi mechanism in plant/fungi, dsRNA/siRNA uptake efficiency, dsRNA/siRNA design parameters, dsRNA stability and delivery strategy, off-target effects, etc. This review emphasizes the significance of these factors and suggests appropriate measures to consider while designing in silico and in vitro experiments for successful RNAi in open-field conditions. We also highlight prospective nanoparticles as smart delivery vehicles for deploying RNAi molecules in plant systems for long-term crop protection and ecosystem compatibility. Lastly, we provide specific directions for future investigations that focus on blending nanotechnology and RNAi-based fungal control for practical applications.

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

Rhizoctonia cerealis is a soilborne fungus that can cause sharp eyespot in wheat, resulting in massive yield losses found in many countries. Due to the lack of resistant cultivars, fungicides have been widely used to control this pathogen. However, chemical control is not environmentally friendly and is costly. Meanwhile, the lack of genetic transformation tools has hindered the functional characterization of virulence genes. In this study, we attempted to characterize the function of virulence genes by two transient methods, host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), which use RNA interference to suppress the pathogenic development. We identified ten secretory orphan genes from the genome. After silencing these ten genes, only the RcOSP1 knocked-down plant significantly inhibited the growth of R. cerealis. We then described RcOSP1 as an effector that could impair wheat biological processes and suppress pathogen-associated molecular pattern-triggered immunity in the infection process. These findings confirm that HIGS and SIGS can be practical tools for researching R. cerealis virulence genes. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In planta RNAi or host-induced gene silencing (HIGS) has undergone significant advancements that have rendered it efficient and stable at the transgenerational level in plants for regulating host genes and targeting genes of insect pests and plant pathogens. Similarly, topical RNAi or spray-induced gene silencing (SIGS) has garnered considerable attention as an environmentally sustainable, selective, and alternative approach to chemical control of insect pests and plant pathogens. Several biotechnology companies and startups have focused their efforts on RNAi-based solutions for topical application in agriculture. Nevertheless, further technological advancements are required to enhance the efficacy of topical RNAi in agriculture, including improved dsRNA delivery systems, better target gene selection, and addressing biosafety regulatory issues. Herein, this review discusses key advances and bottlenecks in RNAi, and summarizes successful applications of these RNAi-based technologies in agriculture focusing on in planta and topical RNAi to control insect pests and plant pathogens. Furthermore, this review delves into the patenting landscape, biosafety considerations, risk evaluations, and the current regulatory status of RNAi in Latin America. Finally, it explores the contributions of RNAi to plant science, food production, and fostering a more sustainable form of agriculture.