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External RNA Application for Gene Regulation and Plant Resistance

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A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Cell Biology".

Deadline for manuscript submissions: closed (15 March 2021) | Viewed by 42336

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Dr. Alexandra S. Dubrovina

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Laboratory of Biotechnology, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Russia
Interests: plant secondary metabolism; RNA interference; plant gene regulation; abiotic stress; transgenic plants; calcium sensor proteins
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Dr. Konstantin V. Kiselev

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Laboratory of Biotechnology, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, 690022 Vladivostok, Russia
Interests: plant secondary metabolism; stilbenes; plant gene expression; plant cell cultures; calcium signaling
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Recent research has shown that small interfering RNAs (siRNAs) and long double-stranded RNA (dsRNAs) externally applied onto the plant or plant pathogen/pest surfaces can be taken up and induce gene silencing. The external RNAs were capable of local and systemic movements inducing RNA interference-mediated plant resistance against invading fungi, viruses, and insects. Recent findings also provided examples of post-transcriptional down-regulation of plant transgenes and endogenes by application of complementary dsRNAs or siRNAs onto the plant surfaces. The exogenously applied RNA molecules were capable of affecting mRNA levels of target genes in the plant genome and in the plant pathogens/pests and, therefore, are promising agents for regulating plant properties.

Further active studies are needed to shed light on the exogenous RNA perception and gene silencing induction in plants, plant pathogens, and pests to promote the development of new innovative approaches in plant biotechnology. This Special Issue will include papers reporting on the effects of externally applied RNAs or providing new knowledge about external RNA perception by plants, plant pathogens, and pests. In addition, this Special Issue welcomes original research papers, reviews, brief reports, communications, and opinion papers focusing on gene silencing induction by external RNAs, RNA stability in the plant environment, and the mechanisms governing RNA trafficking between plants and invading pathogens.

Dr. Alexandra S. Dubrovina
Dr. Konstantin V. Kiselev
Guest Editors

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Keywords

gene regulation
RNA interference
gene silencing
exogenous RNAs
external application
dsRNAs
siRNAs
RNA spraying
RNA stability
plant resistance
plant pathogens

Published Papers (10 papers)

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Research

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33 pages, 48847 KiB

Open AccessArticle

Optimizing Efficient RNAi-Mediated Control of Hemipteran Pests (Psyllids, Leafhoppers, Whitefly): Modified Pyrimidines in dsRNA Triggers

by Wayne Brian Hunter and William M. Wintermantel

Plants 2021, 10(9), 1782; https://doi.org/10.3390/plants10091782 - 26 Aug 2021

Cited by 11 | Viewed by 3723

Abstract

The advantages from exogenously applied RNAi biopesticides have yet to be realized in through commercialization due to inconsistent activity of the dsRNA trigger, and the activity level of RNAi suppression. This has prompted research on improving delivery methods for applying exogenous dsRNA into plants and insects for the management of pests and pathogens. Another aspect to improve RNAi activity is the incorporation of modified 2′-F pyrimidine nucleotides into the dsRNA trigger. Modified dsRNA incorporating 32–55% of the 2′-F- nucleotides produced improved RNAi activity that increased insect mortality by 12–35% greater than non-modified dsRNA triggers of the same sequence. These results were repeatable across multiple Hemiptera: the Asian citrus psyllid (Diaphorina citri, Liviidae); whitefly (Bemisia tabaci, Aleyroididae); and the glassy-winged sharpshooter (Homalodisca vitripennis, Cicadellidae). Studies using siRNA with modified 2′-F- pyrimidines in mammalian cells show they improved resistance to degradation from nucleases, plus result in greater RNAi activity, due to increase concentrations and improved binding affinity to the mRNA target. Successful RNAi biopesticides of the future will be able to increase RNAi repeatability in the field, by incorporating modifications of the dsRNA, such as 2′-F- pyrimidines, that will improve delivery after applied to fruit trees or crop plants, with increased activity after ingestion by insects. Costs of RNA modification have decreased significantly over the past few years such that biopesticides can now compete on pricing with commercial chemical products. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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19 pages, 3751 KiB

Open AccessArticle

Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection

by Maria C. Holeva, Athanasios Sklavounos, Rajendran Rajeswaran, Mikhail M. Pooggin and Andreas E. Voloudakis

Plants 2021, 10(5), 963; https://doi.org/10.3390/plants10050963 - 12 May 2021

Cited by 24 | Viewed by 3918

Abstract

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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21 pages, 407 KiB

Open AccessArticle

RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges

by Daniel Endale Gebremichael, Zeraye Mehari Haile, Francesca Negrini, Silvia Sabbadini, Luca Capriotti, Bruno Mezzetti and Elena Baraldi

Plants 2021, 10(4), 650; https://doi.org/10.3390/plants10040650 - 29 Mar 2021

Cited by 42 | Viewed by 6720

Abstract

Plant pathogenic fungi are the largest group of disease-causing agents on crop plants and represent a persistent and significant threat to agriculture worldwide. Conventional approaches based on the use of pesticides raise social concern for the impact on the environment and human health and alternative control methods are urgently needed. The rapid improvement and extensive implementation of RNA interference (RNAi) technology for various model and non-model organisms has provided the initial framework to adapt this post-transcriptional gene silencing technology for the management of fungal pathogens. Recent studies showed that the exogenous application of double-stranded RNA (dsRNA) molecules on plants targeting fungal growth and virulence-related genes provided disease attenuation of pathogens like Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium graminearum in different hosts. Such results highlight that the exogenous RNAi holds great potential for RNAi-mediated plant pathogenic fungal disease control. Production of dsRNA can be possible by using either in-vitro or in-vivo synthesis. In this review, we describe exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Potential challenges that are faced while developing a RNAi strategy for fungal pathogens, such as off-target and epigenetic effects, with their possible solutions are also discussed. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

16 pages, 3226 KiB

Open AccessArticle

Topical Application of Escherichia coli-Encapsulated dsRNA Induces Resistance in Nicotiana benthamiana to Potato Viruses and Involves RDR6 and Combined Activities of DCL2 and DCL4

by Khouloud Necira, Mongia Makki, Eugenio Sanz-García, Tomás Canto, Fattouma Djilani-Khouadja and Francisco Tenllado

Plants 2021, 10(4), 644; https://doi.org/10.3390/plants10040644 - 29 Mar 2021

Cited by 16 | Viewed by 3287

Abstract

Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants represents an attractive alternative to transgene-based silencing approaches. However, improvement of dsRNA stability in natural conditions is required in order to provide long-term protection against the targeted virus. Here, we tested the protective effect of topical application of Escherichia coli-encapsulated dsRNA compared to naked dsRNA against single and dual infection by Potato virus X expressing the green fluorescent protein (PVX-GFP) and Potato virus Y (PVY) in Nicotiana benthamiana. We found that, in our conditions, the effectiveness of E. coli-encapsulated dsRNA in providing RNAi-mediated protection did not differ from that of naked dsRNA. dsRNA vaccination was partly effective against a dual infection by PVX-GFP and PVY, manifested by a delay in the expression of the synergistic symptoms at early times after inoculation. Using PVX-GFP as a reporter virus together with a suite of RNAi knockdown transgenic lines, we have also shown that RNA-directed RNA polymerase 6 and the combined activities of DICER-like 2 (DCL2) and DCL4 act to promote efficient resistance to virus infection conferred by topical application of dsRNA in N. benthamiana. Our results provide evidence that exogenous dsRNA molecules are processed by the RNA silencing pathways commonly used by the host in response to virus infection. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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13 pages, 1326 KiB

Open AccessArticle

Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana

by Konstantin V. Kiselev, Andrey R. Suprun, Olga A. Aleynova, Zlata V. Ogneva and Alexandra S. Dubrovina

Plants 2021, 10(2), 264; https://doi.org/10.3390/plants10020264 - 30 Jan 2021

Cited by 21 | Viewed by 3983

Abstract

Recent studies have revealed that foliar application of double-stranded RNAs (dsRNAs) or small-interfering RNAs (siRNAs) encoding specific genes of plant pathogens triggered RNA interference (RNAi)-mediated silencing of the gene targets. However, a limited number of reports documented silencing of plant endogenes or transgenes after direct foliar RNA application. This study analyzed the importance of physiological conditions (plant age, time of day, soil moisture, high salinity, heat, and cold stresses) and different dsRNA application means (brush spreading, spraying, infiltration, inoculation, needle injection, and pipetting) for suppression of neomycin phosphotransferase II (NPTII) transgene in Arabidopsis thaliana, as transgenes are more prone to silencing. We observed a higher NPTII suppression when dsRNA was applied at late day period, being most efficient at night, which revealed a diurnal variation in dsRNA treatment efficacy. Exogenous NPTII-dsRNA considerably reduced NPTII expression in 4-week-old plants and only limited it in 2- and 6-week-old plants. In addition, a more discernible NPTII downregulation was detected under low soil moisture conditions. Treatment of adaxial and abaxial leaf surfaces by brushes, spraying, and pipetting showed a higher NPTII suppression, while infiltration and inoculation were less efficient. Thus, appropriate plant age, late time of day, low soil moisture, and optimal dsRNA application modes are important for exogenously induced gene silencing. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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8 pages, 2175 KiB

Open AccessArticle

Plant Functional Genomics in A Few Days: Laser-Assisted Delivery of Double-Stranded RNA to Higher Plants

by Nabil Killiny, Pedro Gonzalez-Blanco, Siddarame Gowda, Xavier Martini and Ed Etxeberria

Plants 2021, 10(1), 93; https://doi.org/10.3390/plants10010093 - 05 Jan 2021

Cited by 7 | Viewed by 2497

Abstract

The technology of transgenic plants is challenging and time consuming, especially for higher plants and trees such as citrus. Double-stranded RNA (dsRNA) delivery via a plant virus is an alternative method to create transgenic plants by suppressing the expression of plant endogenous genes. Citrus tristeza virus-based vector has been constructed specifically for use in citrus trees. However, this is time-consuming, as it can take up to nine months to produce the desired phenotype. Here we describe a much faster method for the study of gene function in citrus trees. In the current study, we used laser light for the delivery of dsRNA to citrus leaves. We targeted the endogenous reporter gene phytoene desaturase (PDS) and obtained the classical phenotype (leaf bleaching) in only three days after the laser-assisted delivery. Interestingly, the phenotype response was systemic, which indicates the movement of dsRNA and/or ssRNA within the plants. In addition, dsRNAs were taken up by phloem cells and the bleaching phenotype was clear around the main veins. In conclusion, the delivery of dsRNA to plants through laser treatment may provide a fast and more specific tool to study the gene function in higher plants and trees. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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Review

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22 pages, 897 KiB

Open AccessReview

RNAs on the Go: Extracellular Transfer in Insects with Promising Prospects for Pest Management

by Dulce Santos, Simon Remans, Stijn Van den Brande and Jozef Vanden Broeck

Plants 2021, 10(3), 484; https://doi.org/10.3390/plants10030484 - 04 Mar 2021

Cited by 5 | Viewed by 3505

Abstract

RNA-mediated pathways form an important regulatory layer of myriad biological processes. In the last decade, the potential of RNA molecules to contribute to the control of agricultural pests has not been disregarded, specifically via the RNA interference (RNAi) mechanism. In fact, several proofs-of-concept have been made in this scope. Furthermore, a novel research field regarding extracellular RNAs and RNA-based intercellular/interorganismal communication is booming. In this article, we review key discoveries concerning extracellular RNAs in insects, insect RNA-based cell-to-cell communication, and plant–insect transfer of RNA. In addition, we overview the molecular mechanisms implicated in this form of communication and discuss future biotechnological prospects, namely from the insect pest-control perspective. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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17 pages, 794 KiB

Open AccessEditor’s ChoiceReview

dsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology

by Nick Wytinck, Christopher L. Manchur, Vivian H. Li, Steve Whyard and Mark F. Belmonte

Plants 2020, 9(12), 1780; https://doi.org/10.3390/plants9121780 - 16 Dec 2020

Cited by 56 | Viewed by 7689

Abstract

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. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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Other

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8 pages, 758 KiB

Open AccessCommentary

Exogenous RNA as a Regulatory Signal during a Plant’s Interaction with the Biotic Environment: An Evolutionary Perspective and Future Applications in Agriculture

by Sergey Ivashuta, Alberto Iandolino and Greg Watson

Plants 2021, 10(3), 532; https://doi.org/10.3390/plants10030532 - 12 Mar 2021

Viewed by 1600

Abstract

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a responsive organism by exogenous RNA. While exogenous RNA transfer between organisms of different kingdoms of life have been unambiguously identified in nature, our understanding of the biological significance of this phenomenon remains obscure, particularly within an evolutionary context. During the last decade multiple reports utilizing various mechanisms of natural eRNAi phenomena have been attempted to develop new agricultural traits and products including weed, disease and insect control. Although these attempts yielded mixed results, this concept remains extremely attractive for many agricultural applications. To better utilize eRNAi for practical applications, we would like to emphasize the necessity of understanding the biological significance of this phenomenon within an evolutionary context and learn from nature by developing advanced tools to identify and study new cases of exogeneous RNA transfer and eRNAi. In this opinion article we would like to look at the exogeneous RNA transfer from an evolutionary perspective, propose that new cases of exogeneous RNA transfer still remain to be identified in nature, and address a knowledge gap in understanding the biological function and significance of RNA transfer. We believe such approach may eventually result in a more successful use of this phenomenon for practical applications in agriculture. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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9 pages, 550 KiB

Open AccessPerspective

Epigenetic Modifications: An Unexplored Facet of Exogenous RNA Application in Plants

by Athanasios Dalakouras and Kalliope K. Papadopoulou

Plants 2020, 9(6), 673; https://doi.org/10.3390/plants9060673 - 26 May 2020

Cited by 19 | Viewed by 3629

Abstract

Exogenous RNA interference (exo-RNAi) is a powerful transgene-free tool in modern crop improvement and protection platforms. In exo-RNAi approaches, double-stranded RNAs (dsRNAs) or short-interfering RNAs (siRNAs) are externally applied in plants in order to selectively trigger degradation of target mRNAs. Yet, the applied dsRNAs may also trigger unintended epigenetic alterations and result in epigenetically modified plants, an issue that has not been sufficiently addressed and which merits more careful consideration. Full article

(This article belongs to the Special Issue External RNA Application for Gene Regulation and Plant Resistance)

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