gmo-free rnai: exogenous application of rna molecules in plants. · 4 40 rnai in plants 41. in...
TRANSCRIPT
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Short title: Exogenous application of RNAi 1
GMO-free RNAi: exogenous application of RNA molecules in 2
plants 3
Athanasios Dalakouras1,2,#
, Michael Wassenegger3, Elena Dadami
1, Ioannis 4
Ganopoulos2, Maria L. Pappas
4 and Kalliope Papadopoulou
1 5
6
1University of Thessaly, Department of Biochemistry & Biotechnology, Larissa, Greece 7
2Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, Thessaloniki, 8
Greece 9
3RLP AgroScience GmbH, AlPlanta Institute for Plant Research, Neustadt, Germany 10
4Democritus University of Thrace, Department of Agricultural Development, Orestiada, 11
Greece 12
# Corresponding author 13
14
Correspondence: 15
Athanasios Dalakouras ([email protected]) 16
University of Thessaly, Department of Biochemistry & Biotechnology, Larissa, Greece, 17
Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, Thessaloniki, 18
Greece 19
One-sentence summary: The latest advances in the field exogenous application of 20
RNA molecules in plants help to protect and modify them through RNA interference 21
(RNAi). 22
Author Contributions: All authors contributed to the writing of this article. 23
Plant Physiology Preview. Published on July 8, 2019, as DOI:10.1104/pp.19.00570
Copyright 2019 by the American Society of Plant Biologists
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Keywords: RNA interference, small RNAs, post-transcriptional gene silencing, RNA-24
directed DNA methylation, high-pressure spraying, trunk injection, petiole absorption. 25
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Summary 26
Since its discovery more than 20 years ago, RNA interference (RNAi) has been 27
extensively used in crop protection platforms. So far, RNAi approaches have been 28
conventionally based on the use of transgenic plants expressing double stranded RNAs 29
(dsRNAs) against selected targets. However, the use of transgenes and genetically 30
modified organisms (GMOs) has raised considerable scientific and public concerns. 31
Hence emerged the need for alternative approaches that avoid the use of transgenes and 32
resort instead to direct exogenous application of RNA molecules that have the potential 33
to trigger RNAi. Here, we highlight the most important advances in this field, 34
discussing the various methods of RNA delivery in plants against diverse targets, such 35
as plant genes, viruses, viroids, fungi, insects, mites, and nematodes. In addition, we 36
examine the possible shortcomings of these methods, underline the critical parameters 37
that have to be met for a desired outcome, and explore feasible possibilities to increase 38
their efficiency and applicability, even against bacterial pathogens. 39
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RNAi in plants 40
In plants, RNA interference (RNAi) is triggered by double stranded RNA (dsRNA) 41
having variable sources of origin, ranging from viral replication intermediates, 42
transcription of inverted repeats, stress-induced overlapping antisense transcripts and 43
RNA-DIRECTED RNA POLYMERASE (RDR) transcription of aberrant transcripts 44
(Hamilton and Baulcombe, 1999; Mette et al., 1999; Borsani et al., 2005; Luo and 45
Chen, 2007). Once present in the plant cell, dsRNAs are processed by DICER-LIKE 46
(DCL) endonucleases into 21-24-nt short interfering RNAs (siRNAs) (Figure 1) (Liu et 47
al., 2009). The model plant Arabidopsis (Arabidopsis thaliana) contains four DCL 48
paralogs. DCL2, DCL3 and DCL4 generate the 22-, 24- and 21-nt siRNAs, 49
respectively, whereas DCL1 recognizes genome-encoded imperfect hairpin RNAs 50
resulting in the biogenesis of 21/22-nt micro RNAs (miRNAs) (Bologna and Voinnet, 51
2014; Borges and Martienssen, 2015). Plant siRNAs and miRNAs, collectively termed 52
small RNAs (sRNAs) (Ruiz-Ferrer and Voinnet, 2009), exhibit 3' 2-nt overhangs and 53
become stabilized though 3’ end methylation by HUE ENHANCER1 (HEN1) (Yu et 54
al., 2005; Yang et al., 2006). After this modification, and depending on its 5´ terminal 55
nucleotide, one of the two sRNA strands will be loaded onto an ARGONAUTE (AGO) 56
protein (Kim, 2008). In general, 21-nt sRNAs with 5’-U are loaded on AGO1 and scan 57
the cytoplasm for complementary transcripts for cleavage and degradation in a process 58
termed post-transcriptional gene silencing (PTGS) (Hamilton and Baulcombe, 1999; Mi 59
et al., 2008) (Figure 1). Twenty-two-nt sRNAs are also loaded on AGO1 but seemingly 60
change AGO1 conformation and recruit RDR6 to the 3’ of the target, transcribing the 61
target transcript into dsRNA and thus generating additional (secondary) siRNAs in a 62
mechanism coined transitive silencing (Dalmay et al., 2000; Chen et al., 2010) (Figure 63
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1). Finally, 24-nt sRNAs with 5’-A are incorporated on AGO4, recognize cognate DNA 64
or its nascent transcript and recruit DNA methyltransferases to methylate the cytosine 65
residues of both DNA strands in a process termed RNA-directed DNA methylation 66
(RdDM) (Wassenegger et al., 1994; Chan et al., 2004) (Figure 1). Importantly, RNAi is 67
not cell-autonomous in plants. Thus, once generated in a single cell, siRNAs are able to 68
move through plasmodesmata to 10-15 neighbouring cells while RNA molecules of a 69
yet unknown nature move through the vasculature system to distant parts of the plant, a 70
phenomenon called systemic silencing (Voinnet and Baulcombe, 1997; Palauqui and 71
Vaucheret, 1998). 72
73
Genetically modified organism (GMO)-free RNAi in plants 74
In addition to mediating a broad range of developmental events, RNAi has great 75
potential against invading pests and pathogens (Eamens et al., 2008; Martinez de Alba 76
et al., 2013). So far, conventional RNAi applications have been largely based on the use 77
of recombinant viruses (virus-induced gene silencing, VIGS), Agrobacterium 78
tumefaciens-mediated transiently expressed transgenes and stably transformed 79
transgenic plants that enable the production of dsRNA molecules against selected 80
targets (host-induced gene silencing, HIGS) (Baulcombe, 2004; Baulcombe, 2015). In 81
2017, the transgenic maize (Zea mays) SmartStax Pro, engineered to expressed dsRNA 82
against corn rootworm (Diabrotica virgivera virgifera), was approved by the United 83
States Environmental Protection Agency, United States Food and Drug Administration 84
and United States Department of Agriculture (https://www.epa.gov/newsreleases/epa-85
registers-innovative-tool-control-corn-rootworm). However, in spite of their 86
demonstrated success, RNAi-based transgenic crops have not been commercialized as 87
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much as one might have expected. Transgenic plants and GMOs in general have met 88
such severe criticism that their chances for widespread approval are gloomy, at least. 89
According to some estimates, it costs approximately 140 million USD to bring a 90
transgenic crop to commercialization (Rosa et al., 2018), and even when that happens, 91
several anti-GMO responses follow. Taking these into consideration, and to tackle this 92
issue, new GMO-free RNAi approaches need to be developed that will enable the 93
activation of RNAi not through the use of recombinant viruses or transgenes but 94
through the direct exogenous delivery of RNA molecules (dsRNAs and/or sRNAs) in 95
plants. In this article, only GMO-free RNAi strategies involving exogenous application 96
of RNA molecules directly in plants will be discussed; transgene-based HIGS and VIGS 97
approaches have been extensively reviewed elsewhere and will not be discussed here 98
(Eamens et al., 2008; Prins et al., 2008; Nowara et al., 2010; Koch and Kogel, 2014; 99
Baulcombe, 2015; Zotti and Smagghe, 2015; Joga et al., 2016; Cai et al., 2018; Rosa et 100
al., 2018; Zotti et al., 2018; Qi et al., 2019). 101
102
Applying RNA molecules in plants to target endogenes and transgenes 103
The first report wherein exogenous RNA application into plants triggered RNAi of a 104
plant gene was described in a 2011 Monsanto patent; Nicotiana benthamiana plants pre-105
treated with Silwet L-77 surfactant and sprayed (2.5 bar) with in vitro transcribed 685 106
bp dsRNA and/or chemically synthesized 21-nt sRNAs targeting the endogenous 107
phytoene desaturase (PDS) mRNA displayed extensive PDS RNAi (Sammons et al., 108
2011). (Note that since chemically synthesized small RNA oligonucleotide duplexes are 109
not the biological outcome of DCL endonucleolytic processing and/or HEN1 110
methylation, they cannot be considered bona fide siRNAs, and thus will be collectively 111
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called hereafter as sRNAs). After this initial observation, several others followed using 112
diverse methods of RNA application, succinctly mentioned below in chronological 113
order. Hence, when Arabidopsis leaves were infiltrated with 21-nt sRNAs fused to a 114
positively charged carrier peptide that combined a copolymer of histidine and lysine, 115
(KH)9 (18 a.a.), RNAi of the yellow fluorescent protein (YFP) transgene and the 116
chalcone synthase (CHS) endogene was recorded (Numata et al., 2014). Subsequent 117
studies demonstrated that RNA molecules can be absorbed by the roots and display 118
biological activity throughout the plant. SHOOT MERISTEMLESS (STM) is a class I 119
knotted-like homeodomain protein expressed in the shoot apical meristem (SAM) and 120
required for SAM formation, while WEREWOLF (WER) is an R2R3-type MyB-related 121
transcription factor expressed in the root epidermal cells. When STM or WER dsRNA 122
conjugated to cationic fluorescent nanoparticle was applied to Arabidopsis seedling 123
roots for five consecutive days, expression of STM and WER was suppressed and 124
resulted in phenotypic defects (Jiang et al., 2014). Moreover, when dsRNA targeting 125
MOB1A and actin was applied to Arabidopsis and rice (Oryza sativa) roots, 126
respectively, RNAi of the corresponding genes took place and normal root growth was 127
perturbed (Li et al., 2015). These results suggest that irrigation-mediated RNA 128
application is a possibility that needs to be further elaborated. In a subsequent study, 129
instead of using in vitro transcribed dsRNA, crude extracts of Escherichia coli HT115 130
(DE3) (see below) expressing a 430 bp dsRNA targeting MYB1 were mechanically 131
inoculated into the Dendrobium hybrida (hybrid orchid) flower buds (Lau et al., 2015). 132
MYB1 is expressed throughout flower bud development and is involved in the 133
development of the conical cell shape of the epidermal cells of the flower labellum. 134
Scanning electron microscopy of the adaxial epidermal cells revealed that application of 135
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MYB1 dsRNA changed the phenotype of floral cells, an outcome of great interest for 136
floriculture biotechnology. 137
Plant cells contain a very tough cellulose-rich cell wall ranging from 0.1 to 138
several µm in thickness, which poses a physical barrier to biomolecule delivery. To 139
facilitate RNA delivery inside the plant cell, RNA molecules are usually conjugated to 140
carrier compounds, as mentioned above (Jiang et al., 2014; Numata et al., 2014). 141
Recently, DNA nanostructures, such as 3D tetrahedron, 1D hairpin tile and 1D 142
nanostring, were used to facilitate the delivery and biological action of 21-nt GFP 143
sRNAs in infiltrated N. benthamiana leaves (Zhang et al., 2019). Instead of remaining 144
in the mesophyllic apoplast, the nanostructure-conjugated sRNAs entered the symplast 145
and silenced GFP expression. Interestingly, sRNAs tethered to 3D nanostructures 146
exhibited both mRNA degradation and translational arrest of the GFP, whereas sRNAs 147
attached to 1D nanostructures led mainly to translational arrest, although the reasons 148
underlying this observation were not elucidated. It should be noted here that, although 149
carrier compounds greatly facilitate RNA delivery, they are also quite expensive and/or 150
difficult to synthesize. The use of carrier compounds seems to be dispensable when 151
plant cell walls are mechanically damaged. Indeed, RNAi of a GFP transgene was 152
efficiently initiated when 22-nt sRNAs or 720 bp dsRNAs (pure nucleic acids) were 153
applied by high-pressure spraying (8 bar) or brush-mediated application on the leaf 154
surface of N. benthamiana and Arabidopsis, respectively (Dalakouras et al., 2016; 155
Dubrovina et al., 2019). 156
157
Transitivity and systemic silencing. 158
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Although 21-nt sRNAs (miRNAs and siRNAs) mediate RNA cleavage in plants, 22-nt 159
sRNAs seemingly recruit RDR6 to the RNA target and are responsible for the 160
amplification, transitive and, likely, systemic spread of RNAi (Chen et al., 2010; 161
Cuperus et al., 2010; McHale et al., 2013). Yet, it had been also suggested that 162
recruitment of RDR6 to their target transcript is mediated not necessarily by 22-nt 163
sRNAs, but by any-sized sRNA that contains an asymmetric bulge in its duplex 164
structure (Manavella et al., 2012). To investigate how the sRNA size/structure affects 165
the onset of local, transitive and systemic RNAi in GFP-expressing N. benthamiana 166
(Nb-GFP) plants, we applied, by high pressure spraying, chemically synthesized 21-, 167
22- and 24-nt GFP sRNAs either as perfect duplexes or as siRNAs containing an 168
asymmetric bulge (Dalakouras et al., 2016). Ultraviolet light examination of sprayed 169
plants revealed that although all tested sRNAs triggered local RNAi to variable degrees, 170
only 22-nt sRNAs (either as perfect bulges or containing an asymmetric bulge) also 171
triggered systemic silencing (Figure 2, left panel). DCL2, responsible for the biogenesis 172
of 22-nt siRNAs, is required for transitivity (Mlotshwa et al., 2008; Parent et al., 2015). 173
Moreover, 22-nt miRNAs recruit RDR6 and trigger transitivity (Chen et al., 2010; 174
Cuperus et al., 2010; McHale et al., 2013). Thus, it is reasonable to assume that any 175
sRNA (siRNA or miRNA or chemically synthesized oligonucleotide) having a size of 176
22 nt can recruit RDR6 to its target and lead to the biogenesis of secondary siRNAs. 177
Upon transitivity, the siRNA population surpasses a certain threshold required for the 178
onset of systemic silencing (Kalantidis et al., 2006; Kalantidis et al., 2008). Thus, 179
transitivity is seemingly connected to generation of systemic silencing signals in the 180
source tissues. Yet, reception of systemic silencing in the sink tissues requires RDR6 181
processing of the target (Schwach et al., 2005). A growing body of recent evidence has 182
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suggested that in contrast to intron-containing genes, intronless genes are much more 183
susceptible to RDR6 processivity and thus transitivity and systemic silencing (Christie 184
and Carroll, 2011; Christie et al., 2011; Dadami et al., 2013; Dadami et al., 2014). 185
Taking these observations into consideration, the chances of systemic RNAi upon 186
exogenous RNA application may significantly increase when then the trigger is 22-nt 187
sRNA and the target gene is intronless (see Outstanding Questions Box). 188
189
Symplastic and apoplastic delivery of RNA 190
It needs to be noted here that depending on the method of RNA application, the 191
efficiency of RNAi fluctuates. When chemically synthesized 22-nt sRNAs were applied 192
in Nb-GFP upon high pressure spraying, they triggered local and systemic RNAi; yet, 193
when they were applied by petiole absorption, they failed to do so (Dalakouras et al., 194
2018). Confocal microscopy revealed that the petiole-absorbed sRNA was transported 195
through the xylem and was restricted to the apoplast (Figure 2, middle panel) 196
(Dalakouras et al., 2018). Similarly, a 499-nt GFP hairpin RNA (hpRNA) applied 197
through petiole absorption and/or trunk injection in grapevine (Vitis vinifera) and apple 198
(Malus domestica) trees was also confined to the xylem and apoplast, and no RNAi was 199
observed (Figure 2, middle and right panel). In summary, when the target of 200
exogenously applied RNA is an intracellular transcript (e.g. plant mRNA or viral/viroid 201
RNA), then the high pressure method ensures efficient RNA delivery into plant cells 202
leading to the onset of, at least, local RNAi. In contrast, RNA applied through petiole 203
absorption and trunk injection is retained in the xylem and apoplast and does not trigger 204
RNAi. However, this is not necessarily a drawback. When dsRNAs are applied to plants 205
with the aim of targeting some insects or fungi (see below), it may be of advantage to 206
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deliver non-processed (by plant DCLs) dsRNAs. Should the plant-applied dsRNAs 207
avoid plant DCL processing, they will be taken up intact by the insects or fungi and be 208
processed therein by the pathogen/pest DICER proteins; the resulting siRNAs will 209
conceivably exhibit greater biological activity against insect or fungal genes. To achieve 210
this, recent approaches rely on the expression of dsRNA transgenes in chloroplasts 211
(Bally et al., 2016; Bally et al., 2018). Chloroplasts lack DCLs and thus the chloroplast-212
expressed dsRNAs remain intact for pathogen/pest uptake. Our own observations 213
suggest that dsRNA application through petiole uptake and/or trunk injection could 214
serve as a GMO-alternative to such transplastomic plants since they both maintain 215
dsRNA intact. 216
217
The issue of epigenetic changes 218
So far, all reports on exogenous applications of dsRNAs in plants aimed to trigger 219
degradation of a given mRNA. However, once present in the plant cell, the applied 220
dsRNAs may be processed not only by DCL4/DCL2 into 21/22-nt siRNAs but also by 221
DCL3 into 24-nt siRNAs, given the DCL co-localization (Pontes et al., 2013; Pumplin 222
et al., 2016). Thus, besides mRNA degradation in the cytoplasm, the applied dsRNAs 223
may also trigger RdDM of the cognate coding region in the nucleus (Dubrovina et al., 224
2019; Dubrovina and Kiselev, 2019). Thus, dsRNA-treated plants considered on the 225
one hand GMO-free, could, on the other hand, be epigenetically modified. Moreover, 226
exogenous application of dsRNA targeting a promoter sequence may trigger not only 227
RdDM but also histone modifications that will eventually result in transcriptional gene 228
silencing (TGS) (Wassenegger, 2005). If RdDM takes place in the dsRNA-treated 229
plants, DOMAINS REARRANGED METHYLTRANSFERASE (DRM2) will mediate 230
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the de novo methylation of the cognate DNA sequence (promoter or coding region) at 231
any sequence context: CG, CHG and CHH (Pelissier et al., 1999; Cao and Jacobsen, 232
2002; Aufsatz et al., 2004). In the progeny of treated plants, CG and, to a lesser extent, 233
CHG methylation can be maintained by METHYLTRANSFERASE 1 (MET1) and 234
CHROMOMETHYLASE 3 (CMT3), respectively (Lindroth et al., 2001; Aufsatz et al., 235
2004). While CG/CHG methylation of a coding region does not seem to negatively 236
affect transcription in plants, CG/CHG methylation of promoter sequences reinforces 237
the heterochromatic state, and thus, TGS can be trans-generationally maintained (Wang 238
et al., 2015; Wakasa et al., 2018). Whether such scenario holds remains to be validated 239
(see Outstanding Questions Box). 240
241
Applying RNA molecules in plants against viruses 242
It has been suggested that RNAi in plants evolved as an antiviral defence mechanism. 243
Indeed, most plant viruses contain single stranded RNA genomes that replicate through 244
dsRNA intermediates. The dsRNA is processed into virus-derived siRNAs resulting in 245
degradation of any homologous RNA, including the single stranded virus RNA 246
genomes. To counter host’s defence, viruses encode RNA silencing suppressors (RSSs), 247
most of which sequester and/or inhibit the function of the siRNAs (Silhavy and 248
Burgyan, 2004; Szittya and Burgyan, 2013). However, the cellular pre-existence of 249
dsRNAs/siRNAs designed to target the virus already before it manages to replicate and 250
generate RSSs is a well-established antiviral crop protection strategy. The GMO 251
approach wherein transgenic plants express dsRNAs against viral proteins has been well 252
documented with very satisfactory results (Prins et al., 2008; Khalid et al., 2017; 253
Pooggin, 2017). Focusing here only on the non-transgenic approaches, Tenllado and 254
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co-workers were the first to demonstrate that exogenous application of dsRNA 255
molecules in plants renders them resistant to viral infections. When pepper mild mottle 256
virus (PMMoV), tobacco etch virus (TEV) and alfalfa mosaic virus (AMV) were 257
mechanically co-inoculated in N. benthamiana leaves with in vitro transcribed 997 bp, 258
1483 bp and 1124 bp dsRNAs targeting the PMMoV replicase, TEV helper component 259
and AMV RNA3, respectively, viral infections were attenuated (Tenllado and Diaz-260
Ruiz, 2001). The authors applied crude extract from E. coli HT115 (DE3) expressing 261
the corresponding dsRNA fragments by lysing cell pellets with a French press and 262
documented similar resistance phenotypes (Tenllado et al., 2003). Using bacterial 263
expression systems, dsRNA expression can easily be scaled-up and still remain cost-264
effective. With an average dsRNA yield of 4 μg per ml of bacterial culture, large 265
fermenters would most possibly meet the huge agricultural demands (Tenllado et al., 266
2004). Besides HT115 (DE3), alternative strains such as M-JM109lacY have been used 267
with equally satisfactory results. Mechanical inoculation of RNA extract from E. coli 268
M-JM109lacY expressing 480 bp dsRNA that target the tobacco mosaic virus (TMV) 269
coat protein (CP) and RNA extract from E. coli HT115 (DE3) expressing 480 bp 270
dsRNA targeting the TMV movement protein (MP) both resulted in viral resistance in 271
tobacco (Nicotiana tabacum) (Yin et al., 2009; Yin et al., 2010). Recently, an 272
alternative system for high quality long dsRNA production was developed, based on 273
Pseudomonas syringae harbouring components of the bacteriophage phi6, wherein 274
dsRNA generation is based not on DNA transcription but on RNA transcription by an 275
RNA-dependent RNA polymerase (RdRP) (Niehl et al., 2018). Compared to E. coli-276
expressed dsRNA, which may result in relatively low yields of fully duplexed dsRNA, 277
the P. syringae system seems advantageous since the RdRP of phage phi6 converts 278
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ssRNA templates into dsRNA with high processivity using a de novo, primer-279
independent initiation mechanism. 280
Using the aforementioned methods, viral resistance has been achieved in a 281
plethora of other cases as listed below: in maize, upon spraying of crude extract from E. 282
coli HT115 (DE3) expressing dsRNA targeting the sugarcane mosaic virus (SCMV) CP 283
(Gan et al., 2010); in papaya (Carica papaya), upon mechanical inoculation of crude 284
extract from E. coli M-JM109lacY expressing 279 bp dsRNA targeting the papaya 285
ringspot virus (PRSV) CP (Shen et al., 2014); in pea (Pisum sativum), upon biolistic 286
delivery of in vitro-transcribed 500 bp dsRNA targeting the pea seed-borne mosaic 287
virus (PSbMV) CP (Safarova et al., 2014); in orchid (Brassolaeliocattleya hybrida), 288
upon mechanical inoculation of crude extract from E. coli HT115 (DE3) expressing 289
dsRNA targeting the cymbidium mosaic virus (CymMV) CP (Lau et al., 2014); in 290
tobacco (N. tabacum), upon mechanical inoculation of in vitro transcribed 237 bp 291
dsRNA targeting the TMV p126 (Konakalla et al., 2016); in cucurbits, upon mechanical 292
inoculation of in vitro transcribed 588 bp dsRNA targeting the zucchini yellow mosaic 293
virus (ZYMV) helper component proteinase (HcPro) (Kaldis et al., 2018); and in N. 294
benthamiana, upon spraying of RNA extracts from P. syringae LM2691 expressing 295
dsRNAs targeting a 2611 bp region of TMV replicase and MP (Niehl et al., 2018). 296
A major issue concerning the aforementioned approaches is that dsRNA 297
application offers a short antiviral protection window (usually 5-10 days), since dsRNA 298
eventually is degraded. Thus, dsRNA would need to be supplied afresh in frequent 299
intervals for lifelong crop protection. To increase the dsRNA stability and thus prolong 300
antiviral protection, Mitter and co-workers bound dsRNA to layered double hydroxide 301
clay nanosheets having an average particle size of 80 to 300 nm (BioClay) (Mitter et al., 302
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2017). The dsRNA bound to BioClay was significantly protected from nucleases, while 303
the dsRNA/BioClay complex did not wash off, even after rigorous rinsing. On the leaf 304
surface, atmospheric CO2 and moisture resulted in a gradual breakdown of BioClay into 305
a biocompatible residue, releasing the dsRNA in the plant cell either by passive 306
diffusion or active transport. A 330 bp dsRNA targeting the CMV CP and bound to 307
BioClay could be detected even 30 days post application (dpa) and upon single 308
application in tobacco resulted in CMV protection for at least 20 days (Mitter et al., 309
2017). Similar results were recently obtained, when spraying of 461 bp dsRNA bound 310
to BioClay and targeting the bean common mosaic virus (BCMV) CP protected N. 311
benthamiana and cowpea (Vigna unguiculata) plants against BCMV infection (Worrall 312
et al., 2019). 313
314
Applying RNA molecules in plants against viroids 315
Viroids are infectious single stranded RNA pathogens that, unlike viruses, are non-316
encapsidated and non-coding but, similar to viruses, trigger the host RNAi mechanism 317
(Tabler and Tsagris, 2004; Tsagris et al., 2008; Flores et al., 2014; Flores et al., 2017). 318
Single-stranded circular RNA viroids exhibit significant intramolecular folding and 319
resemble dsRNA molecules and as such are processed by plant DCLs into siRNAs. 320
Initial reports suggested that viroids are resistant to siRNA-mediated degradation due to 321
their extensive secondary structure (Itaya et al., 2007). Yet, subsequent studies 322
demonstrated that transgenic plants expressing viroid dsRNAs were viroid-resistant 323
(Schwind et al., 2009). Hence, despite their secondary structure, and similar to viruses, 324
viroids can most likely be targeted for sRNA-mediated degradation (Dalakouras et al., 325
2015; Flores et al., 2017). Returning to the non-transgenic approaches, and to the best 326
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of our knowledge, there is only one study where exogenous RNA was applied in plants 327
for anti-viroid resistance, wherein Carbonell and co-workers applied dsRNA in plants 328
against both nuclear-replicating Pospiviroidae and chloroplast-replicating 329
Avsunviroidae members. In tomato (Solanum lycopersicum), gynura (Gynura 330
aurantiaca) and chrysanthemum (Dendranthema grandiflora), mechanical inoculation 331
of in vitro transcribed 353 bp, 364 bp and 399 bp dsRNA targeting regions of Potato 332
spindle tuber viroid (PSTVd), Citrus exocortis viroid (CEVd) and Chrysanthemum 333
chlorotic mottle viroid (CChMVd), respectively, resulted in significant inhibition of 334
infection, at least for up to 30 days dpa (Carbonell et al., 2008). Interestingly, the effect 335
was temperature-dependent, reminiscent of previous reports on the temperature-336
dependency of the general RNAi mechanism in plants (Kalantidis et al., 2002; Szittya et 337
al., 2003). Taking these observations into consideration, exogenous application of RNA 338
in plants for any applications should be performed in a temperature regime of 20-30ο C, 339
since the efficiency of the RNAi machinery is compromised outside this temperature 340
window. 341
342
Applying RNAi molecules in plants against fungi 343
With the exception of Saccharomyces cerevisiae, all fungi, like the rest of eukaryotes, 344
contain several Dicer and Argonaute genes and thus display active RNAi mechanisms. 345
Transgenic plants expressing dsRNAs against fungal genes is a very promising 346
antifungal approach (Koch and Kogel, 2014; Baulcombe, 2015) but will not be 347
addressed here. Koch and co-workers demonstrated that spraying of barley (Hordeum 348
vulgare) with in vitro transcribed 791 bp dsRNA simultaneously targeting three 349
Fusarium graminearum ergosterol biosynthesis genes (CYP51A, CYP51B and 350
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CYP51C) strongly inhibited fungal growth (Koch et al., 2016). Interestingly, the 351
sprayed dsRNA was poorly processed in barley into siRNAs, and the effect on fungal 352
growth was due to the uptaken dsRNA rather than the barley-produced siRNAs, since 353
upon dsRNA spraying no RNAi was observed in a dcl1 F. graminearum mutant. 354
Spraying of in vitro-produced siRNAs also inhibited fungal growth but to lesser extent 355
than dsRNA did. Surprisingly, dsRNA seemed to be more mobile throughout the barley 356
than the smaller siRNAs, but the reasons underlying this phenomenon are unclear. It is 357
also unclear how the dsRNA that first reached the apoplast was transported to the 358
symplast (Koch et al., 2016). In another study, foliar application in Brassica napus of in 359
vitro transcribed dsRNA targeting various fungal genes conferred plant protection 360
against Sclerotinia sclerotium and Botrytis cinerea (McLoughlin et al., 2018). More 361
recently, spraying of in vitro transcribed dsRNA targeting the myosine 5 of Fusarium 362
asiaticum in wounded wheat (Triticum aestivum)oleoptiles resulted in reduced fungal 363
virulence (Song et al., 2018). Yet, the RNAi effect lasted for only 9 hours, unless the 364
dsRNA was continuously supplied. Interestingly, despite the presence of RDR genes in 365
F. asiaticum, small RNA deep sequencing revealed that no secondary siRNAs were 366
generated, suggesting that fungal myosine 5 mRNA could not processed by RDR6. 367
Thus, in contrast to plants where RNAi is maintained through an RDR-mediated self-368
amplification loop, this is likely not happening in fungi. 369
Recent advances from the Hailing Jin laboratory have broadened our knowledge 370
of the mechanistic details of RNA transport phenomena between plants and fungi. 371
Hence, it was discovered that the aggressive fungal pathogen B. cinerea is able not only 372
to deliver siRNAs into host plant cells to suppress host immunity genes, but also uptake 373
exogenously applied dsRNAs and siRNAs that inhibit its growth, providing a typical 374
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case of bi-directional trans-kingdom RNAi (Weiberg et al., 2013; Wang et al., 2016). 375
More specifically, when in vitro-transcribed dsRNA or siRNAs targeting the Bc-DCL1 376
and Bc-DCL2 genes were applied on the surface of fruits (tomato, S. lycopersicum 377
‘Roma’; strawberry, Fragaria × ananassa; and grape, V. labrusca ‘Concord’)), 378
vegetables (iceberg lettuce, Lactuca sativa; and onion, Allium cepa)and flowers (rose, 379
Rosa hybrida) they significantly inhibited grey mould disease (Wang et al., 2016). 380
These findings illustrated that exogenous RNA application can be used to control 381
diseases in post-harvest fruits and vegetables. Whereas the movement of siRNAs 382
through plasmodesmata to neighbouring cells and through the vasculature system to 383
distant parts of the plant is well established, it was unknown until recently how 384
siRNAs/dsRNAs travel across the boundaries between organisms of different 385
taxonomic kingdom, e.g. from plants to fungi and/or vice versa. To investigate how 386
RNA molecules move from plants into interacting fungal cells, Hailing Jin and co-387
workers demonstrated that Arabidopsis secretes exosome-like extracellular vesicles to 388
deliver RNA molecules into B. cinerea (Cai et al., 2018). It is reasonable to assume that 389
such observations reflect a more generalized mechanism of exosome involvement in 390
trans-kingdom RNA transport, and if so, exosomes are likely to be employed in the 391
future in the development of antifungal RNA delivery methods. 392
393
Applying RNAi molecules in plants against insects 394
Triggering RNAi in insects upon exogenous application of dsRNA in plants is a 395
challenging task. Subsequent to the uptake by chewing/sucking insects, the dsRNA 396
must survive the salivary nucleases in the midgut and/or haemolymph, which threaten to 397
quickly degrade it. Next, the dsRNA must be taken up from the epithelial cells through 398
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either the endocytic pathway or the transmembrane Sid-1 channel protein-mediated 399
pathway and processed by Dicer-2 into siRNAs, which will be loaded onto Ago-2 and 400
trigger local RNAi. To be efficiently established throughout the insect, the RNAi 401
molecules (dsRNAs or siRNAs) need to systemically spread to neighbouring and to 402
distant cells. In plants and the nematode Caenorhabditis elegans, systemic spread of 403
RNAi is associated with RNAi amplification by RDRs, proteins that are not present in 404
insects. Yet, despite all these barriers, a proof-of-concept paper demonstrated that 405
western corn rootworm ingestion of vacuolar ATPase dsRNA through artificial diet or 406
upon feeding on dsRNA-expressing transgenic maize plants resulted in significant 407
larval mortality, suggesting that widespread RNAi in insects can be established. Indeed, 408
coleopterans seem to exhibit systemic RNAi spread, although the enzymes involved 409
have not been identified yet (Velez and Fishilevich, 2018). 410
Induction of RNAi in insects through the use of methods such as dsRNA 411
injection, dsRNA-expressing transgenic plants, dsRNA-containing artificial diet or 412
dsRNA-coating of leaf discs has been thoroughly reviewed elsewhere (Zotti and 413
Smagghe, 2015; Joga et al., 2016; San Miguel and Scott, 2016; Bally et al., 2018; Velez 414
and Fishilevich, 2018; Zotti et al., 2018). Exogenous application of dsRNA directly in 415
plants against insects was first demonstrated by Hunter and co-workers, who injected 416
the trunk and/or drenched the roots of citrus and grapevine trees with dsRNA targeting 417
the arginine kinase of two psyllids (Diaphorina citri and Bactericera cockerelli) and the 418
sharpshooter Homalodisca vitripennis. Interestingly, the introduced dsRNA was 419
detected in 2.5 m tall 6-year-old Mexican limes (Citrus aurantifolia) even after 7 weeks 420
post single application (2 g dsRNA diluted in 15 l of water). After this proof-of-concept 421
report, several others followed, exhibiting significant degrees of pest management upon 422
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exogenous application of in vitro transcribed/synthesized RNAs in plants: in Brassica 423
oleracea, upon spraying of 5’-PEG 21-nt siRNAs targeting the diamondback moth 424
Plutella xylostella acetylcholine esterase genes AChE1 and AChE2; in tomato, upon 425
application of dsRNA targeting the Diabrotica virgifera vacuolar ATPase (Ivashuta et 426
al., 2015); in rice, upon root absorption of dsRNA targeting the brown planthopper 427
Nilaparyata lugens P450 (Li et al., 2015); in maize, upon root absorption of dsRNA 428
targeting the Ostrinia furnacalis KTI (Li et al., 2015); in potato (Solanum tuberosum), 429
upon spraying of dsRNA targeting the Colorado potato beetle Leptinosa decemlineata 430
actin; in tomato, upon petiole uptake of dsRNA targeting the tomato leafminer Tuta 431
absoluta vacuolar ATPase (Camargo et al., 2016) and upon root absorption of dsRNA 432
targeting Tuta absoluta ryanodine, acetylcholinesterase and nicotinic acetylcholine 433
alpha 6 (Majidiani et al., 2019). In general, the orders Coleoptera, Lepidoptera and 434
Hemiptera include key pests of crops. Coleopterans are the most susceptible to RNAi, 435
while lepidopterans and hemipterans seem recalcitrant to RNAi, most likely because 436
lepidopterans restrict the up-taken dsRNA to endocytic compartments, and hemipterans 437
inject nucleases into the plant tissue prior to feeding (Lomate and Bonning, 2016; 438
Shukla et al., 2016). Of note, and to improve dsRNA stability, uptake and overall RNAi 439
response, various insecticidal dsRNA formulations have been explored, such as 440
liposomes, chitosan nanoparticles, cationic core-shell nanoparticles and guanylated 441
polymers (Joga et al., 2016; Velez and Fishilevich, 2018). 442
443
Applying RNA molecules in plants against mites 444
Mites belong to the subphylum Chelicerata, a subgroup in the phylum Arthropoda to 445
which insects also belong. Mite genomes encode for most known RNAi enzymes, 446
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21
including two DICERs, seven AGOs and, importantly, five RDRs (Grbic et al., 2011), 447
suggesting that amplification of RNAi and systemic RNAi spread in mites may occur. 448
Indeed, in the citrus red mite Panonychus citri, RNAi spread from the gut to more 449
distant tissues such as cuticle, hemolymph and prothoracic glands (Li et al., 2017). 450
Similar to insects, mites have been targeted for RNAi by various methods, such as 451
feeding on leaves floating on dsRNA solution, dsRNA-expressing transgenic plants and 452
dsRNA-containing artificial diet, as discussed elsewhere (Suzuki et al., 2017; Niu et al., 453
2018). To the best of our knowledge, the only study wherein mites took up dsRNA that 454
was exogenously applied in plants involved mechanical inoculation of tomato leaves 455
with in vitro transcribed dsRNA (targeting the otherwise irrelevant ZYMV HcPro) and 456
the concomitant detection by stem loop RT-PCR of the corresponding siRNAs in the 457
two-spotted spider mite Tetranychus urticae 3 and 10 dpa (Gogoi et al., 2017). 458
However, in that case it was not possible to determine whether the mite absorbed the 459
applied dsRNA or rather the plant-produced siRNAs. In addition, no conclusions could 460
be drawn concerning the RNAi action of the detected siRNAs, since the applied dsRNA 461
had no mite target. In another study where T. urticae was fed on Arabidopsis leaves 462
coated with in vitro transcribed dsRNA targeting the vacuolar ATPase, the mite 463
exhibited visible dark body phenotype but lethality was not observed (Suzuki et al., 464
2017). Interestingly, to identify the most suitable RNAi target genes for mite mortality, 465
two independent studies suggested that coatamer I (COPI) genes and genes involved in 466
the biosynthesis and action of juvenile hormone and action of ecdysteroids were the best 467
candidates (Kwon et al., 2016; Yoon et al., 2018) and should be thus taken into 468
consideration for future RNA applications. 469
470
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Applying RNA molecules in plants against nematodes 471
RNAi was discovered in the model nematode Caenorhabditis elegans (Fire et al., 472
1998). In agriculture, root-knot nematodes cause significant crop losses in several plant 473
species worldwide. Transgenic Arabidopsis expressing 16D10 dsRNA against 474
Meloidogyne incognita, M. javanica, M. arenaria and M. hapla exhibited significant 475
reductions in the number of nematode eggs per gram of root (Huang et al., 2006). So 476
far, no reports on exogenous RNA application in plants against nematodes are available. 477
Arguably, the challenge here would be to deliver RNA molecules to the root tissues. 478
Trunk injection would deliver RNA mainly to the xylem (Dalakouras et al., 2018) and 479
thus only to the upper/aerial part of the plant. Alternatively, RNA molecules can be 480
directly absorbed by the roots (Jiang et al., 2014; Li et al., 2015). It is thus reasonable to 481
assume that RNA molecules conjugated to compounds that would protect them against 482
degradation in the aggressive soil environment could be delivered through irrigation as 483
a nematode protection strategy. 484
485
Perspectives for bacterial management 486
Plant pathogenic bacteria may cause devastating diseases and huge crop losses. 487
Although RNAi is absent in bacteria, similar RNA-based regulation mechanisms do 488
exist in which ~100-nt cis and trans antisense transcripts repress translation and lead to 489
transcript decay (Good and Stach, 2011). Typically, antisense RNAs (asRNAs) that are 490
cis-encoded display high degrees of complementarity with the target mRNA, whereas 491
asRNAs that are trans-encoded exhibit limited complementarity with the target mRNA 492
and require Hfq chaperones to facilitate binding to the target mRNA. So far, exogenous 493
application in plants of synthetic asRNAs designed to combat bacterial pathogens has 494
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23
not been tested. In contrast to dsRNAs applied in RNAi approaches, asRNAs are single 495
stranded and thus more susceptible to plant nucleases. To tackle this problem, 496
exogenously applied asRNAs based on peptide nucleic acids (PNAs) and/or 497
phosphorothioate morpholino oligomers (PMOs) could be used (Good and Stach, 2011). 498
Yet, the biggest challenge upon exogenous application of synthetic asRNAs would be 499
the intracellular delivery across the stringent bacterial cell barriers, since nucleobase 500
oligomers are too large for uptake by simple diffusion. To this end, conjugating the 501
PNA or PMO oligomers to cell cationic entry peptides would greatly facilitate their 502
intracellular delivery (Mellbye et al., 2009). These being said, exogenously applied 503
antibacterial asRNAs could be used with considerable chances of success, particularly 504
in the case of xylem-restricted bacteria such as Xylella fastidiosa, Clavibacter xyli and 505
Pseudomonas syzygii, especially when taking into account that delivery of RNA with 506
robust methods, such as trunk injection or petiole absorption, restricts the presence of 507
the applied RNA to the xylem (Dalakouras et al., 2018) . Besides the aforementioned 508
direct management of bacterial pathogens through exogenous application of synthetic 509
asRNAs, indirect RNAi-based strategies could be applied. For example, although RNAi 510
cannot be used directly against X. fastidiosa, it can nevertheless be employed against its 511
xylem sap-feeding vectors, mainly sharpshooters and spittlebugs (Rosa et al., 2010; 512
Overall and Rebek, 2015). Trunk injected-dsRNAs designed to target essential insect 513
mRNAs will remain unprocessed in the xylem of the woody plant, and provided that 514
they are taken up by the xylem-feeding insect, they will be processed into siRNAs that 515
that will lead to RNAi-mediated insect lethality, ideally minimizing the spread of the 516
devastating pathogen (see Outstanding Questions Box). 517
518
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Concluding Remarks 519
For field-scale management of pests and pathogens, metric ton levels of dsRNAs would 520
be required. A rough estimation suggests 10 g dsRNA per hectare, although this amount 521
may vary depending on the target sensitivity to RNAi and its capacity for systemic 522
RNAi (Zotti et al., 2018). Conceivably, such huge needs could not be merely met by in 523
vitro dsRNA transcription systems, which exhibit a minimum cost of 100 UDS per 1 g 524
of dsRNA. To this end, several industrial companies enable low-cost (almost 2 USD per 525
1 g of dsRNA), large-scale manufacturing of dsRNA (Zotti et al., 2018). According to 526
‘RNAagri’ proprietary technology (https://www.rnagri.com/), bacteria and yeast are 527
engineered to produce the desired dsRNA and a capsid protein which offers protection 528
against nucleases. As the bacteria multiply in the fermentators, the dsRNA:protein 529
complex is self-assembled and accumulates in huge quantities that can be isolated with 530
conventional low-cost methods. The final product is environmentally stable and ready-531
to-use. Field-scale trunk injection of such huge amounts of dsRNAs could be 532
performed by commercially available trunk drilling equipment as the one developed by 533
‘Arborjet’ (https://arborjet.com/arborjet-advantage/). Such advances have facilitated the 534
development of commercial products that are soon to emerge in the market, such as 535
‘BioDirect’, which is designed for insect, virus and weed control and which has already 536
passed the phase I or II of research and development process (Zotti et al., 2018). 537
In summary, exogenous application of RNA molecules with the potential to 538
trigger RNAi is a very powerful tool in modern crop protection and improvement 539
platforms, considering the political and public pressure for sustainable solutions to 540
current agricultural problems. Compared to conventional disease management 541
strategies, exogenous RNAi promises significantly lower off-target effects, since its 542
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25
activity can be narrowed down to a window of few nucleotide complementarity with its 543
target. With the rapidly accumulating sequence datasets, non-conserved regions can be 544
selected as targets to exclude as much as possible off-target effects. To the same end, 545
applying unique sRNAs, rather than long dsRNAs which are processed in a diverse 546
population of siRNAs, would also greatly decrease off-targets. Arguably, the lack of 547
genome data for any existing organisms near the site of exogenous RNA application 548
undermines the proper ecological risk of RNAi technology in the agroecosystem. 549
Nevertheless, the chance to silence non-intended targets is very low and far beyond any 550
other available crop protection strategy has yet to offer. It cannot be excluded that 551
certain pathogens may display gene mutations rendering them resistant to RNAi sprays. 552
In cases of monogenic resistance, alternating ~200 bp regions of the target sequence 553
could tackle this issue, while for polygenic resistance cases, using chimeric RNAs or a 554
mixture of different RNAs could offer a solution, underpinning the overall flexibility of 555
the RNA-based approaches. In the near future, it is reasonable to assume that 556
optimization of RNA production, delivery and stability methods may pave the way for 557
the exogenous application of diverse RNA molecules not necessarily triggering RNAi. 558
Indicatively, such molecules could include: capped and polyadenylated mRNAs when 559
accumulation of a protein is desired; miRNA decoys/sponges for sequestering of a 560
given miRNA and thus upregulating its endogenous target; and even clustered regularly 561
interspaced short palindromic repeats (CRISPR) components (Cas9 mRNA, sgRNAs) 562
for the induction of editing events in a GMO-free manner (see Outstanding Questions 563
Box). 564
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565
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FOOTNOTES 566
AD and KP acknowledge funding from the European Union’s Horizon 2020 research 567
and innovation programme under the Marie Skłodowska-Curie grant agreement N. 568
793186 (RNASTIP). 569
570
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571
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FIGURE LEGENDS 572
573
Figure 1. DCL processing of exogenously applied dsRNA in plants. DCL4 generates 574
21-nt siRNAs that are loaded onto AGO1 and target complementary transcripts for 575
cleavage. DCL2 generates 22-nt siRNAs that are also loaded onto AGO1 and recruit 576
RDR6 to the target transcripts’ 3’ ends for the generation of secondary siRNAs. Finally, 577
DCL3 generates 24-nt siRNAs that are loaded onto AGO4 and trigger de novo DNA 578
methylation by hybridizing with nascent Pol V or Pol II transcripts. Whether DCL1, 579
which is mainly involved in the miRNA pathway, also processes exogenously applied 580
dsRNA is not clear. 581
582
Figure 2. Methods for RNA delivery to plants. Left panel: High-pressure spraying of 583
chemically synthesized GFP sRNAs in GFP-expressing N. benthamiana allows the 584
delivery of sRNAs into the symplast and initiation of RNAi. Only the 22-nt GFP 585
siRNAs, with or without an asymmetric bulge, are able to trigger efficient local (2 dpa) 586
and systemic (20 dpa) RNAi (ultraviolet examination). Notably, targeting the bud 587
instead of the leaf results in more pronounced and widespread RNAi. Middle panel: 588
Petiole absorption of chemically synthesized sRNAs and in vitro transcribed GFP 589
hpRNAs results in the localization of applied RNAs in the apoplast and xylem (confocal 590
microscopy of CY3-labelled RNAs). Thus, the applied 22-nt GFP sRNAs fails to induce 591
RNAi in GFP-expressing N. benthamiana (ultraviolet examination) while the applied 592
GFP hpRNA is not processed by DCLs into GFP siRNAs (Northern blot analysis). 593
Right panel: Trunk injection of in vitro transcribed GFP hpRNA into apple and 594
grapevine results in its localization in the apoplast and xylem. Thus, similar to petiole 595
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30
absorption, no DCL processing takes place (Northern blots). Image adapted from 596
Dalakouras et al., 2016; Dalakouras et al., 2018. 597
598
Figure 3. Exogenous application of RNA molecules into plants against various targets, 599
such as endogenous plant genes, viruses, insects and fungi. In the first two cases, RNAi 600
should take place inside the plant cell. Thus the most suitable application method is 601
high-pressure spraying, which allows symplastic RNA delivery. In contrast, in the cases 602
of insects and fungi, RNAi takes place inside the insect and fungal cells, which thus 603
need to uptake intact dsRNA (unprocessed by the plant DCLs) to achieve efficient 604
RNAi. Hence, in these cases, trunk injection, petiole absorption and/or low-pressure 605
spraying (wherein RNA stays on the leaf surface) are the most suitable methods, since 606
these methods do not result in symplastic RNA delivery. Importantly, while trunk 607
injection and/or petiole absorption is the ideal method for xylem-feeding and/or 608
chewing insects, the spraying method would be more suitable for phloem-feeding 609
insects (e.g. aphids). 610
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958
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ADVANCES
• Exogenous application of RNA molecules (dsRNAs, siRNAs) in plants can trigger RNAi in a GMO-free manner.
• So far, exogenous application of RNA in plants has efficiently triggered RNAi of plant genes, viruses, viroids, fungi, insects and mites.
• Conjugating RNA molecules to nanoparticles and carrier peptides greatly enhances their resistance to nucleases and efficiency of delivery.
• Exogenously applied 22-nt siRNAs are the most potent inducers of local and systemic RNAi in plants.
• High pressure spraying allows the symplastic delivery of exogenous RNA whereas petiole absorption and/or trunk injection results in apoplastic delivery of the exogenous RNA.
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OUTSTANDING QUESTIONS
• Besides RNA degradation, do exogenously applied RNA molecules also trigger epigenetic modifications in plants?
• In contrast to intron-containing genes, are intronless genes more susceptible to systemic RNAi upon exogenous application of RNAs in plants?
• Is the efficiency of RNAi inside insects and fungi higher when they take up dsRNAs that did not undergo processing by plant DCLs?
• Are bacteria susceptible to exogenously applied synthetic asRNAs?
• Besides dsRNAs and siRNAs, could other RNA molecules, such as mRNAs, miRNA sponges and CRISPR components, be efficiently applied in plants?
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DCL4
AGO1 AGO4
DCL2 DCL3
AGO1
AGO1 RDR6
Pol V
CH3
CH3 CH3 CH3
21-nt 22-nt 24-nt Primary siRNAs
dsRNA
RNA RNA DNA
RNA degradation Transitivity DNA methylation
Secondary siRNAs
Figure 1
RDR6
AGO1
CH3 CH3
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2 dpa 20 dpa 30 dpa
sRNA Local RNAi Systemic RNAi
21-nt + -
21-nt asym. + -
22-nt +++ +++
22-nt asym. +++ +++
24-nt - -
24-nt asym. + +
Leaf spraying Bud spraying
HIGH PRESSURE SPRAYING
Applied sRNAs in symplast local and systemic RNAi
PETIOLE ABSORPTION
1 dpa, leaf 1 dpa, petiole 30 dpa
hpRNA siRNAs U6
TRUNK INJECTION
1 3 10 dpa
1 3 10 dpa
hpRNA siRNAs U6
hpRNA siRNAs U6
1 3 10 dpa
Petiole absorption of CY3-labelled 22-nt GFP sRNAs from GFP-expressing N. benthamiana
GFP GFP GFP
GFP GFP GFP
CY3 CY3 GFP
CY3 CY3
Spraying of 22-nt GFP sRNAs in GFP-expressing N. benthamiana
Petiole absorption of CY3-labelled 499-nt GFP hpRNA from N. benthamiana and Northern blots in leaves
Trunk injection of 499-nt GFP hpRNA and Northern blots in leaves
V. vinifera
M. domestica
Applied sRNAs in apoplast/xylem no RNAi
Applied hpRNA in apoplast/xylem no DCL processing no RNAi
Applied hpRNA in apoplast/xylem no DCL processing no RNAi
1 dpa, petiole 1 dpa, stem
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PLANT CELL
Healthy
Insect attack
Viral attack
Fungal attack
dsRNA against plant gene
dsRNA against virus
dsRNA against insect
dsRNA against fungus
INSECT CELL
AAA Plant mRNA
RNAi
PLANT CELL
AAA
RNAi
CAP
VPg Viral RNA
AAA
RNAi
CAP
CAP AAA
nucleus
nucleus
nucleus
nucleus
virus
FUNGAL CELL
Insect mRNA
dsRNA siRNAs
dsRNA siRNAs
dsRNA siRNAs
AAA Fungal mRNA
RNAi
CAP
dsRNA siRNAs
AAA CAP AAA VPg
AAA CAP
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