Journal of Pest Science (2020) 93:1125–1130
https://doi.org/10.1007/s10340-020-01238-2
RAPID COMMUNICATION
RNAi: What is its position in agriculture?
Bruno Mezzetti1 · Guy Smagghe2 · Salvatore Arpaia3 · Olivier Christiaens2 · Antje Dietz‑Pfeilstetter4 ·
Huw Jones5 · Kaloyan Kostov6 · Silvia Sabbadini1 · Hilde‑Gunn Opsahl‑Sorteberg7 · Vera Ventura8 ·
Clauvis Nji Tizi Taning2 · Jeremy Sweet9
Received: 13 March 2020 / Revised: 4 May 2020 / Accepted: 13 May 2020 / Published online: 26 May 2020
© The Author(s) 2020
Abstract
RNA interference (RNAi) is being developed and exploited to improve plants by modifying endogenous gene expression as
well as to target pest and pathogen genes both within plants (i.e. host-induced gene silencing) and/or as topical applications
(e.g. spray-induced gene silencing). RNAi is a natural mechanism which can be exploited to make a major contribution
towards integrated pest management and sustainable agricultural strategies needed worldwide to secure current and future
food production. RNAi plants are being assessed and regulated using existing regulatory frameworks for GMO. However,
there is an urgent need to develop appropriate science-based risk assessment procedures for topical RNAi applications within
existing plant protection products legislation.
Keywords RNAi · dsRNA · Biosafety · Agriculture · Regulations · HIGS · SIGS
Key message
• Plants modified to express target dsRNAs are being
• RNAi is a natural mechanism found in most eukaryotic
assessed and regulated using existing regulatory frameworks for GMO.
• However, there is an urgent need to develop appropriate science-based risk assessment procedures for topical
RNAi applications within existing PPP legislation.
organisms in nature and can be exploited to improve
plant health.
• RNAi-based technology is already being exploited, and
the realized examples confirm its great potential in a
range of areas of crop production and protection.
Communicated by M. Traugott.
* Bruno Mezzetti
b.mezzetti@staff.univpm.it
* Guy Smagghe
guy.smagghe@ugent.be
* Clauvis Nji Tizi Taning
tiziClauvis.taningnji@UGent.be
Jeremy Sweet
jeremysweet303@aol.com
1
Department of Agricultural, Food and Environmental
Sciences, Università Politecnica delle Marche, Ancona, Italy
2
Laboratory of Agrozoology, Department of Plants and Crops,
Faculty of Bioscience Engineering, Ghent University, Ghent,
Belgium
3
DTE-BBC, Italian National Agency for New Technologies,
Energy and Sustainable Economic Development (ENEA),
Rotondella, Italy
4
Institute for Biosafety in Plant Biotechnology, Julius
Kühn-Institut (JKI), Bundesforschungsinstitut für
Kulturpflanzen, Brunswick, Germany
5
IBERS, Aberystwyth University, Aberystwyth, Wales, UK
6
Agrobioinstitute, Agricultural Academy, Sofia, Bulgaria
7
Faculty of Biosciences, Norwegian University of Life
Sciences, Ås, Norway
8
Department of Environmental Science and Policy, Università
degli Studi di Milano, Milan, Italy
9
JT Environmental Consultants Ltd, Cambridge, UK
13
Vol.:(0123456789)
1126
Introduction
Science has taught us about nature’s elegant genetic regulation occurring in eukaryotic organisms like plants and
animals, where double-stranded RNA (dsRNA) molecules
interfere with homologous alien RNA to fine-tune gene
expression and subsequent protein production in a process
called RNA interference (RNAi). Emerging RNAi tools are
increasingly showing potential major impacts on agriculture
with applications in crop protection and production, since
its discovery led to a Nobel Prize in medicine, but with possible applications in other fields of biology (Fire et al. 1991;
Zotti et al. 2018). RNAi is being exploited to adapt endogenous gene expression in plants as well as to target pest and
pathogen genes both within plants (i.e. host-induced gene
silencing, HIGS) and as topical applications (e.g. sprayinduced gene silencing, SIGS). At the molecular level, the
pathway works through processing long dsRNA into socalled small interfering RNA (siRNA) molecules, which
specifically recognize the target messenger RNA (mRNA),
leading to its neutralization. In this way, plant genes can be
targeted to remove unwanted metabolites or increase beneficial nutrients in crops. In pests and pathogens, essential
genes can be suppressed leading to effective protection of
plants. Since siRNAs recognize target gene mRNAs based
on sequence complementarity, systems can be designed with
high specificity where genes with homologous sequences
can be targeted in a narrow range of species. The exponential
increase in available genomic and transcriptomic sequence
data allows the design of highly specific targeting dsRNAs,
minimizing the risk of off-target effects or silencing effects
in non-target organisms (Christiaens et al. 2018).
The importance of RNAi in sustainable
agriculture
Research on a range of potential applications of RNAi in
crop protection is increasing, and it is becoming apparent
that RNAi-based approaches could make a major contribution towards integrated pest management and sustainable agriculture. One of the activities is conducted by the
European COST action “iPlanta” (CA15223)1 was based
on the consideration that, as literature on RNAi-based control in crop protection continues to expand, it is timely to
evaluate both the trends and influence of its development
1
iPlanta is a multi-actor platform of excellence on RNAi mechanisms, applications, biosafety, socioeconomic issues and communication in many EU and nearby countries, and cooperating researchers in
associated countries in North and South America, Australia and Asia.
https://iplanta.univpm.it/
13
Journal of Pest Science (2020) 93:1125–1130
and to provide an indication of the research and development landscape, the prolific centres of research and their
collaborations. Sourcing over 76 million records from the
most comprehensive database, the Thompson Reuters Web
of Science (WoS) using the query string TS = (pest* OR
pathogen* NEAR plant) AND TS = (RNAi OR "RNA interference" OR "RNA-interference"), revealed a rapid global
increase in the number of publications on RNAi research
since year 2002. The top ten countries contributing to RNAi
research span Europe, North America and South-East Asia.
Leaders are in China, USA, India, Germany, Belgium, Japan,
Canada, UK, South Korea and France, with researchers in
China leading the number of publications (Fig. 1). Rapid
developments in RNAi research are led by a diverse set of
collaborative actors from both academia centres and industry, who provide both leadership and globalized contributions to the field across disciplines, space and time (Figs. 2
and 3). Industry pioneers are Devgen N.V. and Monsanto
Co. (now: Bayer CropScience) with their landmark paper
on RNAi to control the western corn rootworm (Baum et al.
2007). Using an alternative plant-mediated RNAi approach,
Mao and co-workers, in another landmark paper, reported
the possibility to control the cotton bollworm, by suppressing its detoxification P450 monooxygenase gene, thereby
impairing its tolerance to gossypol, a natural toxic phytochemical accumulated by plants to resist or evade herbivores
(Mao et al. 2007). The development and number of publications [including patents (see more in Frisio and Ventura
2019)] with RNAi as a tool in crop protection are expected
to keep rising in coming years, supporting further R&D and
implementation in practice.
In the field of plant biotechnology, RNAi has several
unique features which offer additional opportunities to
breeders for varietal improvement compared to genome editing technologies such as CRISPR/Cas or TALENs. One of
these characteristics is that RNAi can lead to a gene knockdown effect, rather than a complete knockout, depending
on the choice of the dsRNA (length and sequence) (Wagner
et al. 2011). This is important when reduced levels of gene
expression are required, as for certain cases of metabolically
engineered plants with modified fatty acid profiles. Other
unique aspects of RNAi are that siRNA molecules have high
mobility through the plant’s vascular system and can move
inside the plant from the point of production to other parts
of the plant (Molnar et al. 2011). Therefore, dsRNA produced in part of the plant (e.g. rootstock or interstock) has
the potential to spread into the grafted parts of the plant so
as to confer resistance to disease to the whole plant, including fruit. This results in fruits that are not genetically modified (GM), but protected by the presence of target-specific
degradable small RNA molecules (De Francesco et al. 2020;
Limera et al. 2017; Zhao and Song 2014).
Journal of Pest Science (2020) 93:1125–1130
1127
Fig. 1 Number of publications in RNAi research disciplines since 2002 and the ten countries with the most authored or co-authored publications
Stable expression of dsRNAs in a GM plant allows exposure to dsRNA by different types of plant feeding arthropods and pathogens in a range of plant tissues as the plant
grows (Zotti et al. 2018). GM plants expressing interfering
RNAs are regulated as other GM plants but are expected
to potentially raise less safety concerns because no new
protein is produced in the plants (Casacuberta et al. 2015;
Ramon et al. 2014) and because of the highly sequencespecific mode of action of RNAi (Tan et al. 2016; Bachman
et al. 2013). In the EU, the EFSA has given biosafety opinions on food and feed for several crops [potato EH92-527-1
(including cultivation in the EU), soybeans MON87705
and MON87705 × MON89788 (excluding cultivation)],
with enhanced nutritional characteristics and more recently
on corn rootworm-resistant maize MON87411 and maize
MON87427 × MON89034 × MIR162 × MON87411 (EFSA
2019). Worldwide, several virus-resistant plants have been
approved for cultivation outside the EU (e.g. plum, squash
and papaya) and many more virus control applications are
being developed (Khalid et al. 2017; Limera et al. 2017). In
addition, plant resistance to a wide range of pests and fungal
pathogens is being studied, particularly to insect vectors of
pathogens and a range of diseases such as cereal rusts or
fruit grey mould (Andrade and Hunter 2016; McLoughlin
et al. 2018; Wang et al. 2016). As with other technologies,
pest and pathogen resistance management is important and
new crop protection applications need to be accompanied
by effective stewardship and resistance management plans.
A more recent innovation is the use of topical applications of dsRNA to induce gene silencing as a new strategy for plant protection or growth regulation (San Miguel
and Scott 2016; Worrall et al. 2019). Technical advances in
the production of dsRNA and formulations to improve the
efficacy, stability and persistence of extracellular dsRNA
mean that it is now realistic to consider using dsRNA for
biological protection (“biopesticide”). It can be applied
as foliar sprays, root drenching, seed treatments or trunk
injections, and there is considerable commercial interest in
this because of the cost of production, the specificity and
improved biosafety compared with chemical pesticides and
some alternative biocontrol strategies (Rodrigues and Petric
2020; Bramlett et al. 2019; Cagliari et al 2019; Zotti et al.
2018). Spray-induced gene silencing (SIGS) is also being
considered for weed control by targeting specific genes in a
weed that do not occur in crops or other weed genera. Such
a strategy would be very useful for controlling grass weeds
in a range of graminaceous crops such as wheat and rice,
though formulations and techniques that allow entry into
weed cells are currently very challenging (Jiang et al. 2014;
Dalakouras et al. 2016).
Topical applications would typically contain dsRNAs
which are produced in microbes or synthesized enzymatically in vitro. Thus, they are not like synthetic agrochemicals
13
1128
Journal of Pest Science (2020) 93:1125–1130
Fig. 2 Global geographical contributions to RNAi research. Leading
countries and key research institutions in RNAi are profiled, indicating a global interest in the field. Records represent the number of
authored and co-authored articles. CA, corresponding author; Univ,
university; Inst, institute
and are different from other biocontrol agents which exploit
proteins such Cry toxins. The dsRNA molecules may be
produced using bacteria and yeasts, but also cell-free mass
production systems are now available. These advances have
lowered the production costs significantly in recent years to
an estimate of 0.5–1 USD per gram, which is now making
RNAi competitive in the market place (Zotti et al. 2018;
Taning et al. 2020). Considering that dsRNA is a natural
biological molecule that is readily degraded in nature and
biological systems, specific formulations to ensure its stability and effective delivery to targets will be required on a
case-by-case basis (Taning et al. 2020). Thus, it represents
a novel type of biological protection/“biopesticide” and it
is important that safety assessments for plant protection
products (PPPs) are adapted to allow introduction of this
technology. Existing PPP risk assessment approaches can be
reliably used to evaluate dsRNA-based products for topical
application, with adaptations only required on a case-bycase basis where additional research might be necessary to
assess risk.
Virus vectors can also be used to enable an efficient
RNAi response in plants and insects. These viruses can be
engineered to contain a fragment of the target gene which
leads to the production of specific dsRNAs in the host cell
(Kurth et al. 2012). The wide range of host-specific viruses
offers an elegant way to modify plant characteristics and to
target insect pests.
Current RNAi-based applications in pest control aim to
kill the target insect pests. However, there is also potential for both HIGS and SIGS to exploit non-lethal modes
of action to result in a more sustainable and integrated
approach to the management of field pests. For example,
when two pheromone-binding proteins were silenced in
the agricultural pest, Helicoverpa armigera by RNAi, male
moths were significantly less able to detect the female sex
pheromone, which reduced mating behaviour (Dong et al.
2017). In another example, RNAi was used to silence spermatogenesis genes in Bactrocera tryoni, a major horticultural pest in Australia, and resulted in dsRNA-treated males
producing 75% fewer viable offspring than negative controls
(Cruz et al. 2018). This opens up the possibility of exploiting RNAi to generate new IPM strategies based on altered
feeding or reproductive behaviour of pests.
13
Journal of Pest Science (2020) 93:1125–1130
1129
Fig. 3 Collaborative patterns
between the 25 most prolific
RNAi research authors. Coauthorship patterns reveal a
general collaborative spirit in
the field. The size of a node
indicates the total number of
records published by the related
author, the weight of an edge
represents the frequency of
the collaborations between the
two authors, and the collaborators listed under each node are
the top three co-authors with
the largest number of copublications. Where only two
collaborators are shown, there
was a multi-way tie for third
Concluding remarks and perspective
In summary, RNAi is a natural mechanism found in most
eukaryotic organisms. RNAi-based technology is already
being exploited, and the marketed products confirm its great
potential in a range of areas of crop production and protection. It can make a major contribution towards integrated
pest management and sustainable agricultural strategies
needed worldwide for current and future food safety and
security. GM RNAi plants are being assessed and regulated
using existing regulatory frameworks. However, there is an
urgent need to adapt existing PPP legislation so that it incorporates appropriate science-based risk assessment procedures for topical RNAi-based applications. This is reflected
in the current activities of the OECD working group on pesticides (OECD 2019).
Looking forward, although Europe is at the forefront of
research on RNAi, the developments and applications may
be constrained by failure of regulators and policymakers in
EU member states to effectively implement current GMO
regulations and by inappropriate and restrictive PPP risk
assessment methods. If this happens, there is likely to be
a disincentive to investment in R&D on agricultural applications of RNAi-based technology in the EU, a declining
trend already attested by the reduction in patent applications. In addition, European farmers will be denied access
to this technology and so lose productivity and competitiveness compared with non-EU countries, just at a time when
sustainable agriculture, integrated pest management and
agricultural biodiversity are in the global spotlight. This will
also result in knock-on effects for consumers, affecting food
availability, choice and price. Thus, policymakers have to
adapt if we are to be part of the solutions.
Acknowledgements The authors acknowledge EU financial support
through iPLANTA COST Action CA 15223. Bruno Mezzetti and
Silvia Sabbadini (UPM) receive funding from the MIUR-PRIN2017
national program via Grant No. 20173LBZM2-Micromolecule, Huw
Jones (IBERS) from the BBSRC via Grant BB/CSP1730/1 and Guy
Smagghe from the Special Research Fund of Ghent University (BOF)
and the Research Foundation—Flanders (FWO). Olivier Christiaens
and Clauvis Nji Tizi Taning are recipient of a postdoctoral fellowship from the Research Foundation—Flanders (FWO) and the Special
Research Fund of Ghent University (BOF), respectively.
Author contributions All authors conceived and wrote the manuscript.
KK made the network analysis and generated the figures. All authors
read, corrected and approved the manuscript.
Funding Not applicable.
Compliance with ethical standards
Conflicts of interest The authors declare no conflicts of interest in relation to this manuscript and state that the opinions expressed are their
own and should not be considered to reflect those of any other individuals or organizations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
13
1130
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
Andrade EC, Hunter WB (2016) RNA interference – natural genebased technology for highly specific pest control (HiSPeC). In:
Abdurakhmonov IY (ed) RNA interference. InTech, Rijeka, pp
391–409
Bachman PM, Bolognesi R, Moar WJ et al (2013) Characterization of
the spectrum of insecticidal activity of a double-stranded RNA
with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte). Transgenic Res 22:1207–1222
Baum JA, Bogaert T, Clinton W et al (2007) Control of coleopteran insect pests through RNA interference. Nat Biotechnol
25:1322–1326
Cagliari D, Dias N, Avila Dos Santos E, Galdeano DM, Smagghe G,
Zotti MJ (2019) Management of pest insects and plant diseases by
non-transformative RNAi. Frontiers Plant Sci 10:1319
Casacuberta JM, Devos Y, Du Jardin P et al (2015) Biotechnological
uses of RNAi in plants: risk assessment considerations. Trends
Biotechnol 33:145–147
Christiaens O, Dzhambazova T, Kostov K, Arpaia S, Joga MR, Urru I,
Sweet J, Smagghe G (1424e) Literature review of baseline information on RNAi to support the environmental risk assessment of
RNAi-based GM plants. EFSA Supp Pub 15:1424e
Cruz C, Tayler A, Whyard S (2018) RNA interference-mediated knockdown of male fertility genes in the Queensland fruit fly Bactrocera
tryoni (Diptera: Tephritidae). Insects 9:96
Dalakouras A, Wassenegger M, McMillan JN, Cardoza V, Maegele I,
Dadami E, Runne M, Krczal G, Wassenegger M (2016) Induction of silencing in plants by high-pressure spraying of in vitrosynthesized small RNAs. Front Plant Sci 7:1327
De Francesco A, Simeone M, Gómez C, Costa N, Garcia ML (2020)
Transgenic Sweet Orange expressing hairpin CP-mRNA in the
interstock confers tolerance to citrus psorosis virus in the nontransgenic scion. Transgenic Res 29:215
Dong K, Sun L, Liu JT et al (2017) RNAi-induced electrophysiological
and behavioral changes reveal two pheromone binding proteins of
Helicoverpa armigera involved in the perception of the main sex
pheromone component Z11–16: Ald. J Chem Ecol 43:207–214
Fire A, Albertson D, Harrison S, Moerman D (1991) Production of
antisense RNA leads to effective and specific inhibition of gene
expression in C. elegans muscle. Development 113:503–514
Frisio DG, Ventura V (2019) Exploring the Patent Landscape of
RNAi-based Innovation for Plant Breeding. Recent Pat Biotechnol 13:207–216
EFSA Panel on Genetically Modified Organisms (GMO), Naegeli
H, Bresson JL et al (2019) Assessment of genetically modified
maize MON 87427× MON 89034× MIR 162× MON 87411 and
subcombinations, for food and feed uses, under Regulation (EC)
No 1829/2003 (application EFSA‐GMO‐NL‐2017‐144). EFSA
J 17:e05848
Jiang L, Ding L, He B, Shen J, Xu Z, Yin M, Zhang X (2014) Systemic
gene silencing in plants triggered by fluorescent nanoparticledelivered double-stranded RNA. Nanoscale 6:9965–9969
13
Journal of Pest Science (2020) 93:1125–1130
Khalid A, Zhang Q, Yasir M, Li F (2017) Small RNA based genetic
engineering for plant viral resistance: application in crop protection. Front Microbiol 8:43
Kurth EG, Peremyslov VV, Prokhnevsky AI, Kasschau KD, Miller M,
Carrington CC, Dolya VV (2012) Virus-derived gene expression
and RNA interference vector for grapevine. J Virol 86:6002–6009
Limera C, Sabbadini S, Sweet JB, Mezzetti B (2017) New biotechnological tools for the genetic improvement of major woody fruit
species. Front Plant Sci 8:1418
Mao YB, Cai WJ, Wang JW et al (2007) Silencing a cotton bollworm
P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25:1307–1313
McLoughlin AG, Walker PL, Wytinck N, Sullivan DS, Whyard S, Belmonte MF (2018) Developing new RNA interference technologies
to control fungal pathogens. Can J Plant Pathol 40:325–335
Molnar A, Melnyk C, Baulcombe DC (2011) Silencing signals in
plants: a long journey for small RNAs. Genome Biol 12:215
Bramlett M, Plaetinck G, Maienfisch P (2019) RNA-Based Biocontrols—a New Paradigm in Crop Protection. Engineering. https://
doi.org/10.1016/j.eng.2019.09.008
OECD (2019) OECD Conference on RNAi based pesticides. https://
www.oecd.org/chemicalsafety/pesticides-biocides/conference-onrnai-based-pesticides.htm
Ramon M, Devos Y, Lanzoni A et al (2014) RNAi-based GM
plants: food for thought for risk assessors. Plant Biotechnol J
12:1271–1273
Rodrigues TB, Petrick JS (2020) Safety Considerations for humans and
other vertebrates to agricultural uses of externally applied RNA
molecules. Front Plant Sci 11:407
San Miguel K, Scott JG (2016) The next generation of insecticides:
DsRNA is stable as a foliar applied insecticide. Pest Manag Sci
72:801–809
Tan J, Levine SL, Bachman PM et al (2016) No impact of DvSnf7 RNA
on honey bee (Apis mellifera L.) adults and larvae in dietary feeding tests. Environ Toxicology Chem 35:287–294
Taning CNT, Arpaia S, Christiaens O et al (2020) RNA-based biocontrol compounds: current status and perspectives to reach the
market. Pest Manag Sci 76:841–845
Wagner N, Mroczka A, Roberts PD, Schreckengost W, Voelker T
(2011) RNAi trigger fragment truncation attenuates soybean
FAD2-1 transcript suppression and yields intermediate oil phenotypes. Plant Biotechnol J 9:723–728
Wang M, Weiberg A, Lin F-M, Thomma BPHJ, Huang H-D, Jin H
(2016) Bidirectional cross kingdom RNAi and fungal uptake of
external RNAs confer plant protection. Nat Plants 2:16151
Worrall EA, Bravo-Cazar A, Nilon AT, Fletcher SJ, Robinson KE,
Carr JP, Mitter N (2019) Exogenous application of RNAi-inducing
double-stranded RNA inhibits aphid-mediated transmission of a
plant virus. Front Plant Sci 10:265
Zhao D, Song GQ (2014) Rootstock-to-scion transfer of transgenederived small interfering RNA s and their effect on virus
resistance in nontransgenic sweet cherry. Plant Biotechnol J
12:1319–1328
Zotti M, dos Santos EA, Cagliari D, Christiaens O, Taning CNT,
Smagghe G (2018) RNAi technology in crop protection against
arthropod pests, pathogens and nematodes. Pest Manag Sci
74:1239–2125
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.