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RNA Interference-Based Tools for Plant Improvement and Protection 2.0

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (31 August 2023) | Viewed by 20292

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Special Issue Editors

Dr. Alexandra S. Dubrovina

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Guest Editor

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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Guest Editor

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,

RNA interference (RNAi) is a conserved intracellular process that mediates plant resistance to undesirable nucleic acids and regulates expression of endogenous protein-coding genes. This naturally occurring phenomenon is used to considerably downregulate expression of specific gene targets and induce desirable biochemical and/or phenotype changes in plants or plant pathogens. The major RNAi-based crop improvement and protection strategies include host-induced gene silencing (HIGS), virus-induced gene silencing (VIGS), feeding insects with double-stranded RNA (dsRNA), and external dsRNA application termed as spray-induced gene silencing (SIGS) or exogenous RNAi (exo-RNAi). Recent research has shown that double-stranded RNA (dsRNAs), small interfering RNAs (siRNAs), and microRNA (miRNA) exogenously applied on plant surfaces can be taken up and induce gene silencing in plants and plant pathogens. External RNA application is being developed as an innovative approach for gene regulation in plants and plant pathogens. All these techniques present promising instruments for ensuring low-risk and environmentally friendly plant management.

This Special Issue invites original research papers, reviews, communications, and opinion papers devoted to the application of RNAi-based technologies for plant improvement, gene function analysis, or plant pathogen/pest control.

We welcome papers related but not limited to the following specific topics:

Exogenous RNAs for gene regulation, plant improvement, gene function analysis, or plant pathogen/pest control;
Host-induced gene silencing (HIGS): recent achievements and developments;
Virus-induced gene silencing (VIGS): recent achievements and developments;
Insect dsRNA feeding and foliar dsRNA application to control insect pests;
Bacterium-mediated RNAi;
Other innovative RNAi-based techniques for plant gene regulation;
RNAi as a tool for plant gene function analysis;
Stability and/or uptake of exogenous dsRNA;
Mechanisms of exogenous RNA perception and RNAi induction in plants and plant pathogens.

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

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Keywords

RNA interference
gene regulation
exogenous RNA application
spray-induced gene silencing (SIGS)
host-induced gene silencing (HIGS)
virus-induced gene silencing (VIGS)
siRNA
dsRNA
crop improvement
plant pathogen control

Published Papers (9 papers)

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Research

Jump to: Review

17 pages, 3640 KiB

Open AccessArticle

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

by Viktoriya O. Samarskaya, Nadezhda Spechenkova, Irina Ilina, Tatiana P. Suprunova, Natalia O. Kalinina, Andrew J. Love and Michael E. Taliansky

Int. J. Mol. Sci. 2023, 24(21), 15769; https://doi.org/10.3390/ijms242115769 - 30 Oct 2023

Cited by 1 | Viewed by 1127

Abstract

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

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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21 pages, 1715 KiB

Open AccessArticle

Transgenic East African Highland Banana Plants Are Protected against Radopholus similis through Host-Delivered RNAi

by Henry Shaykins Mwaka, Lander Bauters, Josephine Namaganda, Shirley Marcou, Priver Namanya Bwesigye, Jerome Kubiriba, Guy Smagghe, Wilberforce Kateera Tushemereirwe and Godelieve Gheysen

Int. J. Mol. Sci. 2023, 24(15), 12126; https://doi.org/10.3390/ijms241512126 - 28 Jul 2023

Cited by 1 | Viewed by 903

Abstract

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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13 pages, 2418 KiB

Open AccessArticle

Exogenously Induced Silencing of Four MYB Transcription Repressor Genes and Activation of Anthocyanin Accumulation in Solanum lycopersicum

by Andrey R. Suprun, Konstantin V. Kiselev and Alexandra S. Dubrovina

Int. J. Mol. Sci. 2023, 24(11), 9344; https://doi.org/10.3390/ijms24119344 - 26 May 2023

Cited by 1 | Viewed by 1266

Abstract

RNA interference (RNAi) is a natural post-transcriptional regulatory mechanism that can be artificially induced by exogenous application of double-stranded RNAs (dsRNAs) to the plant surfaces. Recent studies show that it is possible to silence plant genes and change plant properties using plant RNA spraying and other approaches for dsRNA delivery. In this study, we investigated the effect of exogenous gene-specific dsRNAs on the silencing of four tomato genes encoding MYB-family transcription repressors of anthocyanin biosynthesis in the leaves of tomato Solanum lycopersicum L. We found that the exogenous application of dsRNAs encoding for the SlMYBATV1, SlMYB32, SlMYB76, and SlTRY genes downregulated mRNA levels of these endogenous repressors of anthocyanin production, upregulated the expression of anthocyanin biosynthesis-related genes, and enhanced anthocyanin content in the leaves of S. lycopersicum. The data demonstrated that exogenous gene-specific dsRNAs can induce post-transcriptional gene silencing in tomato leaves by direct foliar application of dsRNAs. This approach may be used for plant secondary metabolism induction and as a silencing tool for gene function studies without the need to produce genetically modified plants. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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26 pages, 2670 KiB

Open AccessArticle

Nanoparticle-Shielded dsRNA Delivery for Enhancing RNAi Efficiency in Cotton Spotted Bollworm Earias vittella (Lepidoptera: Nolidae)

by Shelja Sandal, Satnam Singh, Gulshan Bansal, Ramandeep Kaur, Kanakachari Mogilicherla, Suneet Pandher, Amit Roy, Gurmeet Kaur, Pankaj Rathore and Anu Kalia

Int. J. Mol. Sci. 2023, 24(11), 9161; https://doi.org/10.3390/ijms24119161 - 23 May 2023

Cited by 7 | Viewed by 2065

Abstract

The spotted bollworm Earias vittella (Lepidoptera: Nolidae) is a polyphagous pest with enormous economic significance, primarily affecting cotton and okra. However, the lack of gene sequence information on this pest has a significant constraint on molecular investigations and the formulation of superior pest management strategies. An RNA-seq-based transcriptome study was conducted to alleviate such limitations, and de novo assembly was performed to obtain transcript sequences of this pest. Reference gene identification across E. vittella developmental stages and RNAi treatments were conducted using its sequence information, which resulted in identifying transcription elongation factor (TEF), V-type proton ATPase (V-ATPase), and Glyceraldehyde -3-phosphate dehydrogenase (GAPDH) as the most suitable reference genes for normalization in RT-qPCR-based gene expression studies. The present study also identified important developmental, RNAi pathway, and RNAi target genes and performed life-stage developmental expression analysis using RT-qPCR to select the optimal targets for RNAi. We found that naked dsRNA degradation in the E. vittella hemolymph is the primary reason for poor RNAi. A total of six genes including Juvenile hormone methyl transferase (JHAMT), Chitin synthase (CHS), Aminopeptidase (AMN), Cadherin (CAD), Alpha-amylase (AMY), and V-type proton ATPase (V-ATPase) were selected and knocked down significantly with three different nanoparticles encapsulated dsRNA conjugates, i.e., Chitosan-dsRNA, carbon quantum dots-dsRNA (CQD-dsRNA), and Lipofectamine-dsRNA conjugate. These results demonstrate that feeding nanoparticle-shielded dsRNA silences target genes and suggests that nanoparticle-based RNAi can efficiently manage this pest. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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17 pages, 4100 KiB

Open AccessArticle

Effect of Gene Silencing of Translation Initiation Factors eIF(iso)4G and eIF(iso)4E on Sour Cherry Rootstock Resistance to Sharka Disease

by Lilia Mourenets, Alexander Pushin, Vadim Timerbaev, Tatyana Khmelnitskaya, Eduard Gribkov, Nikita Andreev and Sergey Dolgov

Int. J. Mol. Sci. 2023, 24(1), 360; https://doi.org/10.3390/ijms24010360 - 26 Dec 2022

Cited by 1 | Viewed by 1604

Abstract

Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line’s plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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Review

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18 pages, 1253 KiB

Open AccessReview

RNA Interference Past and Future Applications in Plants

by Sarah Koeppe, Lawrence Kawchuk and Melanie Kalischuk

Int. J. Mol. Sci. 2023, 24(11), 9755; https://doi.org/10.3390/ijms24119755 - 05 Jun 2023

Cited by 8 | Viewed by 4216

Abstract

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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25 pages, 5078 KiB

Open AccessReview

RNAi Technology: A New Path for the Research and Management of Obligate Biotrophic Phytopathogenic Fungi

by Isabel Padilla-Roji, Laura Ruiz-Jiménez, Nisrine Bakhat, Alejandra Vielba-Fernández, Alejandro Pérez-García and Dolores Fernández-Ortuño

Int. J. Mol. Sci. 2023, 24(10), 9082; https://doi.org/10.3390/ijms24109082 - 22 May 2023

Cited by 3 | Viewed by 2478

Abstract

Powdery mildew and rust fungi are major agricultural problems affecting many economically important crops and causing significant yield losses. These fungi are obligate biotrophic parasites that are completely dependent on their hosts for growth and reproduction. Biotrophy in these fungi is determined by the presence of haustoria, specialized fungal cells that are responsible for nutrient uptake and molecular dialogue with the host, a fact that undoubtedly complicates their study under laboratory conditions, especially in terms of genetic manipulation. RNA interference (RNAi) is the biological process of suppressing the expression of a target gene through double-stranded RNA that induces mRNA degradation. RNAi technology has revolutionized the study of these obligate biotrophic fungi by enabling the analysis of gene function in these fungal. More importantly, RNAi technology has opened new perspectives for the management of powdery mildew and rust diseases, first through the stable expression of RNAi constructs in transgenic plants and, more recently, through the non-transgenic approach called spray-induced gene silencing (SIGS). In this review, the impact of RNAi technology on the research and management of powdery mildew and rust fungi will be addressed. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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26 pages, 817 KiB

Open AccessReview

Role of Plant Virus Movement Proteins in Suppression of Host RNAi Defense

by Anastasia K. Atabekova, Anna D. Solovieva, Denis A. Chergintsev, Andrey G. Solovyev and Sergey Y. Morozov

Int. J. Mol. Sci. 2023, 24(10), 9049; https://doi.org/10.3390/ijms24109049 - 20 May 2023

Cited by 3 | Viewed by 1919

Abstract

One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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22 pages, 2451 KiB

Open AccessReview

Current Scenario of Exogenously Induced RNAi for Lepidopteran Agricultural Pest Control: From dsRNA Design to Topical Application

by Vívian S. Lucena-Leandro, Emanuel F. A. Abreu, Leonardo A. Vidal, Caroline R. Torres, Camila I. C. V. F. Junqueira, Juliana Dantas and Érika V. S. Albuquerque

Int. J. Mol. Sci. 2022, 23(24), 15836; https://doi.org/10.3390/ijms232415836 - 13 Dec 2022

Cited by 4 | Viewed by 3425

Abstract

Invasive insects cost the global economy around USD 70 billion per year. Moreover, increasing agricultural insect pests raise concerns about global food security constraining and infestation rising after climate changes. Current agricultural pest management largely relies on plant breeding—with or without transgenes—and chemical pesticides. Both approaches face serious technological obsolescence in the field due to plant resistance breakdown or development of insecticide resistance. The need for new modes of action (MoA) for managing crop health is growing each year, driven by market demands to reduce economic losses and by consumer demand for phytosanitary measures. The disabling of pest genes through sequence-specific expression silencing is a promising tool in the development of environmentally-friendly and safe biopesticides. The specificity conferred by long dsRNA-base solutions helps minimize effects on off-target genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera through non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to innovative solutions. Considerations about the regulation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives have revealed a worthy effort to control Lepidoptera pests with this new mode of action, which provides more sustainable and reliable technologies for field management. New data on the genomics of this taxon may contribute to a future customized target gene portfolio. As a case study, we illustrate how dsRNA and associated methodologies could be applied to control an important lepidopteran coffee pest. Full article

(This article belongs to the Special Issue RNA Interference-Based Tools for Plant Improvement and Protection 2.0)

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