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RNAi-mediated resistance to viruses: a critical assessment of methodologies
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ScienceDirect RNAi-mediated resistance to viruses: a critical assessment of methodologies Mikhail M Pooggin In plants, RNA interference (RNAi)-based antiviral defense is mediated by multigenic families of Dicer-like enzymes generating small interfering (si)RNAs from double-stranded RNA (dsRNA) produced during replication and/or transcription of RNA and DNA viruses, and Argonaute enzymes binding viral siRNAs and targeting viral RNA and DNA for siRNA-directed posttranscriptional and transcriptional silencing. Successful viruses are able to suppress or evade the production or action of viral siRNAs. In antiviral biotech approaches based on RNAi, transgenic expression or non-transgenic delivery of dsRNA cognate to a target virus pre-activates or boosts the natural plant antiviral defenses. Design of more effective antiviral RNAi strategies requires better understanding of viral siRNA biogenesis and viral anti-silencing strategies in virus-infected plants.
Address INRA, UMR BGPI, Montpellier, France Corresponding author: Pooggin, Mikhail M ([email protected])
Current Opinion in Virology 2017, 26:28–35 This review comes from a themed issue on Engineering for viral resistance Edited by John Carr and Peter Palukaitis For a complete overview see the Issue and the Editorial Available online 25th July 2017 http://dx.doi.org/10.1016/j.coviro.2017.07.010 1879-6257/ã 2017 Elsevier B.V. All rights reserved.
Introduction A pathogen-derived resistance (PDR) approach, in which functional or dysfunctional pathogen sequences are transferred to a host genome or delivered transiently in a host cell, has proven to be applicable for protecting plants against viruses. The first evidence for an RNA-mediated mechanism underlying PDR was obtained in transgenic plants expressing untranslatable sense or antisense forms of viral coat protein gene sequences [1,2]. The mechanism was post-transcriptional and highly sequencespecific: no broad-spectrum virus resistance was observed. Some of the transgenic lines displayed symptoms but eventually recovered from viral disease. The highly resistant and recovery phenotypes and the reduction of steady-state RNA levels in recovered transgenic leaf Current Opinion in Virology 2017, 26:28–35
tissues were proposed to be manifestations of a common mechanism [1,2]. This mechanism was initially called RNA-mediated virus resistance or posttranscriptional gene silencing (PTGS). Now it is broadly recognized as RNA silencing which also includes RNA-mediated transcriptional gene silencing (TGS) and RNA interference (RNAi) [3].
The molecular mechanism underlying RNA silencing phenomena Studies of transgene/endogene co-suppression phenomena and antiviral responses in plants have led to the discoveries of small (s)RNAs associated with co-suppression and RNA virus infection [4], RNA-directed DNA methylation associated with TGS [5] and RNA viroid infection [6], and systemic silencing likely mediated by phloem-mobile sRNAs [7,8]. Parallel studies of RNAi phenomena in invertebrate animals and fungi revealed that RNAi is triggered by double-stranded RNA (dsRNA) [9], Dicer processes dsRNA into sRNAs [10], Argonaute (AGO)/slicer forms an RNA-induced silencing complex (RISC) mediating sRNAdirected cleavage of complementary target mRNA [11], and RNA-dependent RNA polymerase (RDR) [12] generates dsRNA precursors of secondary sRNAs. All these components of the RNA silencing machinery have subsequently been identified in plants which possess multigenic families encoding Dicer-like (DCL), AGO and RDR enzymes with specialized and partially redundant functions in regulation of plant development, physiology and stress responses as well as in defenses against transposons, transgenes, viruses and non-viral pathogens [13,14]. Plant sRNAs are classified into microRNAs (miRNAs) produced by DCLs from hairpin-like structures of MIR gene transcripts and small interfering RNAs (siRNAs) produced by DCLs from RDR-dependent and RDR-independent dsRNA precursors originating from multiple genes/ genomic loci and viruses. Both miRNAs and siRNAs are sorted by AGOs, mostly based on sRNA size and 50 -nucleotide (nt) identity, to form RISCs mediating PTGS via sRNA-directed mRNA cleavage or translational repression and TGS via sRNA-directed DNA methylation [13,15]. The earlier observations that viruses are both inducers and targets of RNA silencing and that plants infected with one virus can become resistant to closely related viruses/ strains [16] can now be explained by the sequencespecific action of viral siRNAs. The antiviral RNAi-based biotech methods rely on pre-activation of the plant RNA silencing machinery by transgenic expression or transient application of dsRNA cognate to a target virus. Such www.sciencedirect.com
Transgenic RNAi-mediated resistance to plant viruses Pooggin 29
dsRNA are processed into siRNAs, similar to natural dsRNA precursors of viral siRNAs, to confer PTGS and/or TGS-mediated resistance against the target virus and related viruses sharing sufficient sequence identity. Below I describe the biogenesis of viral siRNAs and the viral anti-silencing strategies in virus-infected plants, and then focus on the design of antiviral RNAi constructs and their application in transgenic plants. More comprehensive reviews have previously described various types of RNA-mediated virus resistance and protein-based PDR [17–19].
Production and action of viral siRNAs in virus-infected plants
different steps of the biogenesis and/or action of viral siRNAs as well as plant miRNAs [34]. One of the common strategies evolved by RNA viruses involves the binding of sRNAs by a viral suppressor protein, preventing RISC assembly [35]. This strategy may account for large quantities of viral siRNAs accumulating in some virus-infected plants without any apparent antiviral effect. Furthermore, viruses can evade silencing by evolving the protective secondary structures that prevent RISC access and by expressing from highly structured regions the decoy dsRNAs that engage the silencing machinery in massive siRNA production and thereby protect other regions of the virus genome from repressive siRNAs [24,33,36,37].
In all virus-infected plants the RNA silencing machinery generates siRNAs from the entire virus genome sequence in both sense and antisense polarities. Indeed, complete genomes of DNA and RNA viruses and viroids can be reconstructed by siRNA sequencing and de novo assembly [20]. Nonetheless, viral sequences are targeted differentially, resulting in siRNA hotspots [20]. The hotspot profile and sequence features of viral siRNAs, which reflect relative plant defense and viral counter-defense activities, should be taken into consideration for better design of antiviral RNAi constructs.
Design of antiviral RNAi constructs
The genetic requirements for biogenesis and action of viral siRNAs have been thoroughly investigated in Arabidopsis and, to a lesser extent, in Nicotiana benthamiana and rice. All four Arabidopsis DCLs are involved in the generation of viral siRNAs [21], but their impact on antiviral defense depends on the infecting virus. RNA viruses with cytoplasmic replication/transcription are targeted mostly by DCL4 and DCL2 generating 21-nt and 22-nt siRNAs [21,22,23], while DNA viruses with nuclear replication and/or transcription are, in addition to DCL4 and DCL2, targeted by DCL3 and DCL1 generating 24-nt and 21-nt siRNAs, respectively [21,24,25]. Similarly, RDR1, RDR2 and RDR6 are involved in defense against RNA viruses and production of viral secondary siRNAs [26–28], while neither RDR1, RDR2 nor RDR6 contribute to the biogenesis of DNA virus-derived siRNAs [24,25]. Among multiple AGOs implicated in plant antiviral defense [29], AGO2 preferentially binding 21–22-nt 50 A-RNAs plays a major role, owing to its ability to bind viral siRNAs [28,30] and form RISCs with cleavage/slicer activity [31], while AGO1 binding 21–22-nt 50 U-RNAs plays both direct and indirect roles via its association with viral siRNAs [28,32] or plant miRNAs [30], respectively. AGO4 binding 24-nt 50 A-RNAs has been implicated in defense against DNA viruses, although its association with 24-nt viral siRNAs was not confirmed [24,33].
The originally designed hairpin RNAs contained a spacer between the inverted repeats [39,42]. Replacement of the spacer with an intron increased antiviral immunity [39]. Intron excision may help to align the complementary hairpin arms, promoting the formation of a duplex. Alternatively, splicing may facilitate the hairpin’s passage from the nucleus, or create a smaller, less nucleasesensitive loop. However, neither a bigger loop size nor an unpaired 50 -extention interfered with hairpin RNAmediated silencing [43]. Thus, the spicing event itself is important, likely because it is coupled with nuclear export of hairpin RNA.
To counteract these antiviral defenses, most plant viruses have evolved silencing suppressor proteins targeting www.sciencedirect.com
Concurrent with the discovery that dsRNA is a trigger of RNAi in animals [9], simultaneous expression of sense and antisense RNAs in transgenic plants was found to be a more potent inducer of antiviral silencing than separate expression of sense or antisense RNAs [38]. This finding paved a way for subsequent design of RNAi constructs expressing an intron-spliced hairpin RNA with inverted repeats of a viral sequence and their successful application against RNA and DNA viruses as well as viroids [39,40,41] (Figure 1a).
In RNAi-transgenic tomato immune to tomato yellow leaf curl virus (TYLCV, DNA begomovirus) [44], a 200-nt intron RNA accumulated at high steady state levels, while the hairpin RNA composed of 700-nt arms was fully processed into highly-abundant 21–22 nt siRNAs and less abundant 24-nt siRNAs, as demonstrated by sRNA sequencing combined with blot hybridization [45]. These findings together with previous characterization of genetic requirements for the biogenesis of intronhairpin RNA -derived siRNAs in Arabidopsis [23] suggest that the hairpin RNA transcript is efficiently spliced and transported to the cytoplasm where it is processed by DCL4 and DCL2 into 21-nt and 22-nt siRNAs, respectively, while a small proportion of hairpin RNA is retained in the nucleus and processed by DCL3 into 24-nt siRNAs (Figure 1a). Analysis of 50 -nucleotide identities of the transgenic siRNAs showed their possible association with Current Opinion in Virology 2017, 26:28–35
30 Engineering for viral resistance
Figure 1
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The model for biogenesis and the single-nucleotide resolution maps of intron-hairpin RNA transgene-derived siRNAs. (a) The transgene is shown schematically with the promoter, the inverted repeat and the intervening intron boxed. Pol II generates a capped and polyadenylated transcript that folds back to form a duplex. The resulting hairpin undergoes splicing and nuclear export. A fraction of unspliced hairpin is processed by DCL3 into 24-nt siRNAs mediating phloem-mobile silencing, whereas the spiced hairpin is processed by DCL4 and DCL2 into 21-nt and 22-nt siRNAs mediating cell-autonomous silencing of the target virus. (b) Small RNAs from virus-free RNAi-transgenic tomato plants, sequenced and mapped to the transgene cassette reference sequence as describes in [45]. The transgene cassette is shown schematically with the 35S promoter, the TYLCV Rep gene inverted repeats, the intervening intron and the terminator boxed. The histograms plot the number of 21-nt, 22-nt, and 24-nt siRNA reads at each nucleotide position of the transgene expression cassette in sense (bars above the axis) and antisense (bars above the axis) orientation. Note that the mapping tool distributed identical reads equally (50–50%) between the two inverted repeat sequences, resulting in mirroring profiles of the siRNA hotspots. The read number scales are different between more abundant 21–22-nt siRNAs and less abundant 24-nt siRNAs.
multiple AGOs including the antiviral AGO1, AGO2 and AGO4 [45]. Moreover, the transgenic siRNAs accumulated at much higher levels (6–8% of total sRNAs) than TYLCV viral siRNAs in non-transgenic plants (2–3%), thus explaining their durable protective effect [44,45].
did not result in antiviral immunity [49,50]. In those cases where hairpin RNA transgenes failed to exert antiviral effects, quantities and size/50 -nucleotide/hotspot profiles of transgenic siRNAs were not analyzed by deep sequencing.
It has been reported that hairpin RNA transcription does not necessarily guarantee triggering antiviral RNAi [46]. However, a spacer-hairpin RNA investigated in that study would be inefficiently transported to the cytoplasm for DCL4-mediated and DCL2-mediated production of siRNAs targeting a cytoplasmic RNA virus [46]. The higher accumulation levels of hairpin RNA transgenederived siRNAs generally correlated with the higher resistance to both RNA and DNA viruses [42,47,48]. Nonetheless, in some cases transgenic siRNA production
Besides Fuentes et al. [45], several studies have applied deep sequencing to characterize intron-hairpin RNAderived siRNAs in transgenic plants resistant to RNA and DNA viruses [51,52,53,54–56]. Notably the hotspot profiles of transgenic siRNAs were generally quite similar to those of virus target sequence-derived viral siRNAs, and the quantities of transgenic siRNAs exceeded those of viral siRNAs in non-transgenic plants, thus supporting the notion that hairpin RNA transgenes pre-activate and boost the natural antiviral mechanism.
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Transgenic RNAi-mediated resistance to plant viruses Pooggin 31
Leibman et al. [51] reported that the intron-hairpin RNA transgenic cucumber line with higher siRNA quantity (42% of total sRNAs) exhibited broad-spectrum resistance to both target and non-target RNA potyviruses, compared to the transgenic line with lower siRNA quantity (13%) exhibiting resistance only to the target potyvirus. A notable difference between the two lines is that the former had an increased RDR1 expression and accumulated 24-nt transgenic siRNAs, whereas the latter had no RDR1 induction and accumulated only 21–22 nt siRNAs [51], suggesting a contribution of RDR1 and/or 24-nt siRNAs to broader resistance. Zhao and Song [52] reported that RNAi-transgenic cherry plants resistant to an RNA ilarvirus accumulated siRNAs of low abundance (0.2% of total sRNAs), but the predominance of 24-nt and 21-nt classes with a strong bias to 50 A and 50 U, respectively, could explain the protectiveness of low abundance siRNAs. Moreover, the follow-up study demonstrated mobility of the 24-nt siRNAs from the transgenic cherry rootstock to a non-transgenic scion, which conferred antiviral resistance in the grafted scions [53]. The mobility of a small proportion of intron-hairpin RNA-derived siRNAs from the transgenic rootstock to a non-transgenic scion was also reported in tomato plants. However, only 21–22-nt siRNAs were found in the grafted scions, since 24-nt siRNAs were not expressed in the rootstock, and the scions were susceptible to target begomovirus infection [55]. Thus, both cellautonomous 21–22-nt siRNAs and mobile 24-nt siRNAs contribute to virus resistance in RNAi-transgenic plants (Figure 1a). This is consistent with the findings that mobile 24-nt sRNAs can direct epigenetic silencing in Arabidopsis [7,57]. The sRNA sequencing also revealed that the production of intron-hairpin RNA-derived siRNAs is restricted to the viral inverted repeats [45,54] (Figure 1b). Thus, secondary siRNA production outside of the hairpin arms (i.e. promoter, intron and terminator), which may result in transgene silencing, is inefficient: this explains robust siRNA expression and durable antiviral immunity of intron-hairpin RNA-transgenic plants for several generations under field conditions [45].
The choice of an RNAi target region within a virus genome RNAi constructs with different lengths of a hairpin arm/ viral target sequence have been successfully used in various plant species [18]. Both short (50–150 bp) and longer (up to 2.5 kbp; [58]) hairpins were protective. More diverse siRNA species derived from longer hairpins are expected to be more protective as they target longer viral sequences. However they may also cause off-target effects, that is, siRNA-directed silencing of host plant gene(s) with a negative impact on plant development or physiology. To avoid off-target effects, artificial miRNA transgenes designed to express a 21–22 nt miRNA-like www.sciencedirect.com
species cognate to a target virus have been employed [59]. However, viruses quickly evolve to mutate a short target sequence and thereby evade the artificial miRNAmediated resistance in transgenic plants [60,61]. Moreover, artificial miRNA-mediated virus resistance was shown to be non-transmissible through graft junction [62], possibly due to the lack of mobile 24-nt siRNAs. This issue is important for woody perennial plants where grafting of non-transgenic scions on transgenic rootstocks is considered as the most promising antiviral strategy [63]. The choice of target sequences within a virus genome can be important to achieve resistance against both RNA and DNA viruses. Indeed, transgenic RNAi resistance to segmented RNA viruses in rice required identification and targeting of a viral ‘Achilles’ heel’ gene [64]. The reason why some of the viral target genes/genome regions are resilient to transgenic siRNAs was not systematically investigated. It is conceivable that GC-rich target sequences would tend to form secondary structures, preventing RISC access, while AU-poor hairpin arms would generate low abundance 50 A-siRNA and 50 U-siRNA, reducing the chances to form active RISCs with AGO1, AGO2 and/or AGO4. It is also conceivable that viral genome regions that do not spawn abundant siRNAs in virus-infected plants are more optimal targets than viral siRNA hotspot regions which might deliberately produce dsRNA decoys [24,37]. Consistent with the latter hypothesis, mutations in the siRNA hotspot sequences within an RNAi target region did not break RNAi-mediated antiviral immunity [51], indicating that the transgenic siRNAs targeting non-hotspot viral sequences are sufficiently protective. In the case of DNA begomoviruses, targeting a bidirectional promoter region by intron-hairpin RNA resulted in recovery from initial virus infection in transiently transformed mungbean seedlings [40] and transgenic cassava plants [65], whereas targeting an AC1/Rep coding sequence resulted in complete antiviral immunity in transgenic cassava [48], tomato [44] and common bean [66] plants. In the latter cases, the transgenic siRNAs likely mediated both posttranscriptional silencing of the viral Rep mRNA and transcriptional silencing of the AC2 gene whose promoter elements are located in the upstream AC1 target sequence.
How to achieve broader and more durable antiviral resistance through RNAi? Because of sequence specificity of RNAi, the spectrum of virus resistance is generally restricted to viral strains or closely related viruses with greater than ca. 90% sequence identities. The broader spectrum resistance could be achieved through transgenic expression of hairpin RNAs with chimeric arms composed of sequences from two or more viruses [47,58,67,68], or cassettes composed of Current Opinion in Virology 2017, 26:28–35
32 Engineering for viral resistance
several separate intron-hairpin RNAs targeting different viruses [69]. The durability of RNAi-mediated resistance may depend on relative abilities of target viruses to overcome RNAi. Indeed, viral strains encoding stronger silencing suppressors could in some cases brake RNAi-mediated resistance [70]. Likewise, a non-target virus can potentially suppress RNAi resistance to a target virus. Furthermore, a target virus can potentially mutate the RNAi target sequence or replace it by recombination with a distantly related virus, as was recently reported for RNAi-transgenic tomato plants under open field conditions [45]: some of the asymptomatic transgenic plants contained a chimeric virus, in which a large region of the targeted monopartite begomovirus containing the 700 nt RNAi target sequence was replaced with a homologous region from a non-target bipartite begomovirus (Figure 2). The recombinant virus evaded the repressive action of transgenic siRNAs, because it shares only 62% homology with the hairpin arm sequence (with no stretch of nucleotide identity longer than 17 nts) [45].
Concluding remarks Despite numerous reports on successful application of transgenic RNAi against economically important plant
viruses, field trials and commercialization of RNAi-transgenic plants were carried out only in a few cases, likely because of negative public perception of genetically modified organisms. Recently, intron-hairpin RNA-transgenic common bean plants resistant to bean golden mosaic virus [66] have been accepted for commercial use in Brazil: these plants exhibited durable virus resistance under open field conditions as well as unaltered agronomic characteristics and nutritional value [71,72,73]. Other examples of transgenic crops approved for commercial use mostly include those in which virus resistance was mediated by expression of viral coat protein genes [19]. One of the concerns is that constitutive siRNA expression (under control of CaMV 35S promoter used in most RNAi transgenes) would lead to off-target effects. Indeed, the transcriptome profiling of two RNAi-transgenic tomato lines with different selection markers revealed a common set of differentially expressed genes [45]. It is not clear, however, whether the transgenic siRNAs had direct or indirect effects. Interestingly, the co-regulated genes did not include any silencing-related genes, suggesting the plant silencing machinery was not affected or saturated by the production of abundant intron-hairpin RNA-derived siRNAs [45].
Figure 2
Parental monopartite target begomovirus
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Recombination-dependent evasion of antiviral RNAi in transgenic tomato plants resistant to tomato leaf curl virus (TYLCV) under open field conditions. The intron-hairpin RNA-transgenic plants were immune to TYLCV disease transmitted to all control tomato plants by viruliferous whiteflies from neighboring virus-infected plants. Molecular analysis revealed that some of the asymptomatic transgenic lines contained a recombinant virus in which a large region of the TYLCV genome (AC1/AC4/AC2/AC3 genes) was replaced with the corresponding region from an unknown virus distantly-related to two known bipartite begomoviruses. The recombinant virus, named Tomato latent virus (TLV), evaded transgenic RNAi, because an RNAi target region between AC4 and AC2 genes was only 62% identical to that of TYLCV (for details, see [45]). Current Opinion in Virology 2017, 26:28–35
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Transgenic RNAi-mediated resistance to plant viruses Pooggin 33
As an alternative to transgenic RNAi, non-transgenic delivery of dsRNA or siRNAs to plant tissues has been considered, since topical dsRNA application was shown to confer resistance against plant viruses [74]. This approach is still hampered by transient protective effects and high costs of dsRNA synthesis, although cost-effective methods of dsRNA production have been developed [75] and delivery methods are being improved to stabilize dsRNA and thereby prolong the antiviral RNAi effect [76].
14. Zvereva AS, Pooggin MM: Silencing and innate immunity in plant defense against viral and non-viral pathogens. Viruses 2012, 4:2578-2597.
Conflict of interest
19. Khalid A, Zhang Q, Yasir M, Li F: Small RNA based genetic engineering for plant viral resistance: application in crop protection. Front Microbiol 2017, 8:43.
The author declares no conflict of interest.
Acknowledgements I thank Alejandro Fuentes for fruitful collaboration on RNAi-transgenic tomato plants. The work was supported by Swiss National Science Foundation [grant N 155737].
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37. Rajeswaran R, Golyaev V, Seguin J, Zvereva AS, Farinelli L, Pooggin MM: Interactions of Rice tungro bacilliform pararetrovirus and its protein P4 with plant RNA-silencing machinery. Mol Plant Microbe Interact 2014, 27:1370-1378. The dsRNA decoy mechanism of RNAi evasion is conserved in pararetroviruses of monocot and dicot plants.
51. Leibman D, Wolf D, Saharan V, Zelcer A, Arazi T, Yoel S, Gaba V, Gal-On A: A high level of transgenic viral small RNA is associated with broad potyvirus resistance in cucurbits. Mol Plant Microbe Interact 2011, 24:1220-1238. The first study that applied sRNA sequencing analysis of intron-hairpin RNA transgene-derived siRNAs, and showed broader-spectrum resistance associated with higher 24-nt siRNA and RDR1 levels and irrelevance of targeting viral siRNA hotspots.
38. Waterhouse PM, Graham MW, Wang MB: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci U S A 1998, 95:13959-13964.
52. Zhao D, Song GQ: High-throughput sequencing as an effective approach in profiling small RNAs derived from a hairpin RNA expression vector in woody plants. Plant Sci 2014, 228:39-47.
39. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Nature 2000, 407:319-320. The first report of successful application of intron-hairpin RNA transgenes to confer plant immunity to an RNA virus. 40. Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T: RNAi targeting of DNA virus in plants. Nat Biotechnol 2003, 21:131 132. The first report showing that transient expression of intron-hairpin RNA transgenes can cure plants from DNA virus infection. 41. Adkar-Purushothama CR, Kasai A, Sugawara K, Yamamoto H, Yamazaki Y, He YH, Takada N, Goto H, Shindo S, Harada T, Sano T: RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains. Sci Rep 2015, 5:17949.
53. Zhao D, Song GQ: Rootstock-to-scion transfer of transgene derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry. Plant Biotechnol J 2014, 12:1319-1328. The first evidence that phloem-mobile 24-nt siRNAs derived from intronhairpin RNAi transgene contribute to virus resistance in non-transgenic tissues. 54. Montes C, Castro A´, Barba P, Rubio J, Sa´nchez E, Carvajal D, Aguirre C, Tapia E, DelI´ Orto P, Decroocq V, Prieto H: Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge. Virus Genes 2014, 49:325-338. 55. Leibman D, Prakash S, Wolf D, Zelcer A, Anfoka G, Haviv S, Brumin M, Gaba V, Arazi T, Lapidot M, Gal-On A: Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA. Arch Virol 2015, 160:2727-2739.
42. Kalantidis K, Psaradakis S, Tabler M, Tsagris M: The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact 2002, 15:826-833.
56. Wang F, Li W, Zhu J, Fan F, Wang J, Zhong W, Wang MB, Liu Q, Zhu QH, Zhou T et al.: Hairpin RNA targeting multiple viral genes confers strong resistance to rice black-streaked dwarf virus. Int J Mol Sci 2016, 17:E705.
43. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA et al.: Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 2001, 27:581-590. Analysis of parameters for optimal design of RNAi constructs targeting plant genes and RNA virus.
57. Lewsey MG, Hardcastle TJ, Melnyk CW, Molnar A, Valli A, Urich MA, Nery JR, Baulcombe DC, Ecker JR: Mobile small RNAs regulate genome-wide DNA methylation. Proc Natl Acad Sci U S A 2016, 113:E801-E810.
44. Fuentes A, Ramos PL, Fiallo E, Callard D, Sa´nchez Y, Peral R, Rodrı´guez R, Pujol M: Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to tomato yellow leaf curl virus infection in transgenic tomato plants. Transgenic Res 2006, 15:291-304. 45. Fuentes A, Carlos N, Ruiz Y, Callard D, Sa´nchez Y, Ochagavı´a ME, Seguin J, Malpica-Lo´pez N, Hohn T, Lecca MR et al.: Field trial and molecular characterization of RNAi-transgenic tomato Current Opinion in Virology 2017, 26:28–35
58. Liu Z, Scorza R, Hily J-M, Scott SW, James D: Engineering resistance to multiple Prunus viruses through expression of chimeric hairpins. J Am Soc Horticult Sci 2007, 132:407-414. 59. Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH: Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 2006, 24:14201428. 60. Simo´n-Mateo C, Garcı´a JA: MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily www.sciencedirect.com
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evolves to escape this silencing mechanism. J Virol 2006, 80:2429-2436. 61. Martı´nez F, Lafforgue G, Morelli MJ, Gonza´lez-Candelas F, Chua NH, Daro`s JA, Elena SF: Ultradeep sequencing analysis of population dynamics of virus escape mutants in RNAimediated resistant plants. Mol Biol Evol 2012, 29:3297-3307. 62. Zhang X, Li H, Zhang J, Zhang C, Gong P, Ziaf K, Xiao F, Ye Z: Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner. Transgenic Res 2011, 20:569-581. 63. Cirilli M, Geuna F, Babini AR, Bozhkova V, Catalano L, Cavagna B, Dallot S, Decroocq V, Dondini L, Foschi S et al.: Fighting sharka in peach: current limitations and future perspectives. Front Plant Sci 2016, 7:1290. 64. Sasaya T, Nakazono-Nagaoka E, Saika H, Aoki H, Hiraguri A, Netsu O, Uehara-Ichiki T, Onuki M, Toki S, Saito K, Yatou O: Transgenic strategies to confer resistance against viruses in rice plants. Front Microbiol 2014, 4:409. 65. Vanderschuren H, Akbergenov R, Pooggin MM, Hohn T, Gruissem W, Zhang P: Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs. Plant Mol Biol 2007, 64:549-557. 66. Bonfim K, Faria JC, Nogueira EO, Mendes EA, Araga˜o FJ: RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact 2007, 20:717-726. 67. Arif M, Azhar U, Arshad M, Zafar Y, Mansoor S, Asad S: Engineering broad-spectrum resistance against RNA viruses in potato. Transgenic Res 2012, 21:303-311. 68. Chung BN, Yoon JY, Palukaitis P: Engineered resistance in potato against potato leafroll virus, potato virus A and potato virus Y. Virus Genes 2013, 47:86-92.
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69. Zhang X, Sato S, Ye X, Dorrance AE, Morris TJ, Clemente TE, Qu F: Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. Phytopathology 2011, 101:1264-1269. 70. Hassani-Mehraban A, Brenkman AB, van den Broek NJ, Goldbach R, Kormelink R: RNAi-mediated transgenic Tospovirus resistance broken by intraspecies silencing suppressor protein complementation. Mol Plant Microbe Interact 2009, 22:1250-1257. 71. Araga˜o FJ, Faria JC: First transgenic geminivirus-resistant plant in the field. Nat Biotechnol 2009, 27:1086-1088. The first open field trial and agronomic evaluation of the intron-hairpin RNA transgenic tomato line resistant to tomato yellow leaf curl disease, which led to its approval for commercial use and human consumption. 72. Araga˜o FJ, Nogueira EO, Tinoco ML, Faria JC: Molecular characterization of the first commercial transgenic common bean immune to the Bean golden mosaic virus. J Biotechnol 2013, 166:42-50. 73. Carvalho JL, de Oliveira Santos J, Conte C, Pacheco S, Nogueira EO, Souza TL, Faria JC, Araga˜o FJ: Comparative analysis of nutritional compositions of transgenic RNAimediated virus-resistant bean (event EMB-PV051-1) with its non-transgenic counterpart. Transgenic Res 2015, 24:813-819. 74. Tenllado F, Dı´az-Ruı´z JR: Double-stranded RNA-mediated interference with plant virus infection. J Virol 2001, 75:1228812297. 75. Voloudakis AE, Holeva MC, Sarin LP, Bamford DH, Vargas M, Poranen MM, Tenllado F: Efficient double-stranded RNA production methods for utilization in plant virus control. Methods Mol Biol 2015, 1236:255-274. 76. Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, Fletcher SJ, Carroll BJ, Lu GQ, Xu ZP: Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants 2017, 3:16207.
Current Opinion in Virology 2017, 26:28–35
ScienceDirect RNAi-mediated resistance to viruses: a critical assessment of methodologies Mikhail M Pooggin In plants, RNA interference (RNAi)-based antiviral defense is mediated by multigenic families of Dicer-like enzymes generating small interfering (si)RNAs from double-stranded RNA (dsRNA) produced during replication and/or transcription of RNA and DNA viruses, and Argonaute enzymes binding viral siRNAs and targeting viral RNA and DNA for siRNA-directed posttranscriptional and transcriptional silencing. Successful viruses are able to suppress or evade the production or action of viral siRNAs. In antiviral biotech approaches based on RNAi, transgenic expression or non-transgenic delivery of dsRNA cognate to a target virus pre-activates or boosts the natural plant antiviral defenses. Design of more effective antiviral RNAi strategies requires better understanding of viral siRNA biogenesis and viral anti-silencing strategies in virus-infected plants.
Address INRA, UMR BGPI, Montpellier, France Corresponding author: Pooggin, Mikhail M ([email protected])
Current Opinion in Virology 2017, 26:28–35 This review comes from a themed issue on Engineering for viral resistance Edited by John Carr and Peter Palukaitis For a complete overview see the Issue and the Editorial Available online 25th July 2017 http://dx.doi.org/10.1016/j.coviro.2017.07.010 1879-6257/ã 2017 Elsevier B.V. All rights reserved.
Introduction A pathogen-derived resistance (PDR) approach, in which functional or dysfunctional pathogen sequences are transferred to a host genome or delivered transiently in a host cell, has proven to be applicable for protecting plants against viruses. The first evidence for an RNA-mediated mechanism underlying PDR was obtained in transgenic plants expressing untranslatable sense or antisense forms of viral coat protein gene sequences [1,2]. The mechanism was post-transcriptional and highly sequencespecific: no broad-spectrum virus resistance was observed. Some of the transgenic lines displayed symptoms but eventually recovered from viral disease. The highly resistant and recovery phenotypes and the reduction of steady-state RNA levels in recovered transgenic leaf Current Opinion in Virology 2017, 26:28–35
tissues were proposed to be manifestations of a common mechanism [1,2]. This mechanism was initially called RNA-mediated virus resistance or posttranscriptional gene silencing (PTGS). Now it is broadly recognized as RNA silencing which also includes RNA-mediated transcriptional gene silencing (TGS) and RNA interference (RNAi) [3].
The molecular mechanism underlying RNA silencing phenomena Studies of transgene/endogene co-suppression phenomena and antiviral responses in plants have led to the discoveries of small (s)RNAs associated with co-suppression and RNA virus infection [4], RNA-directed DNA methylation associated with TGS [5] and RNA viroid infection [6], and systemic silencing likely mediated by phloem-mobile sRNAs [7,8]. Parallel studies of RNAi phenomena in invertebrate animals and fungi revealed that RNAi is triggered by double-stranded RNA (dsRNA) [9], Dicer processes dsRNA into sRNAs [10], Argonaute (AGO)/slicer forms an RNA-induced silencing complex (RISC) mediating sRNAdirected cleavage of complementary target mRNA [11], and RNA-dependent RNA polymerase (RDR) [12] generates dsRNA precursors of secondary sRNAs. All these components of the RNA silencing machinery have subsequently been identified in plants which possess multigenic families encoding Dicer-like (DCL), AGO and RDR enzymes with specialized and partially redundant functions in regulation of plant development, physiology and stress responses as well as in defenses against transposons, transgenes, viruses and non-viral pathogens [13,14]. Plant sRNAs are classified into microRNAs (miRNAs) produced by DCLs from hairpin-like structures of MIR gene transcripts and small interfering RNAs (siRNAs) produced by DCLs from RDR-dependent and RDR-independent dsRNA precursors originating from multiple genes/ genomic loci and viruses. Both miRNAs and siRNAs are sorted by AGOs, mostly based on sRNA size and 50 -nucleotide (nt) identity, to form RISCs mediating PTGS via sRNA-directed mRNA cleavage or translational repression and TGS via sRNA-directed DNA methylation [13,15]. The earlier observations that viruses are both inducers and targets of RNA silencing and that plants infected with one virus can become resistant to closely related viruses/ strains [16] can now be explained by the sequencespecific action of viral siRNAs. The antiviral RNAi-based biotech methods rely on pre-activation of the plant RNA silencing machinery by transgenic expression or transient application of dsRNA cognate to a target virus. Such www.sciencedirect.com
Transgenic RNAi-mediated resistance to plant viruses Pooggin 29
dsRNA are processed into siRNAs, similar to natural dsRNA precursors of viral siRNAs, to confer PTGS and/or TGS-mediated resistance against the target virus and related viruses sharing sufficient sequence identity. Below I describe the biogenesis of viral siRNAs and the viral anti-silencing strategies in virus-infected plants, and then focus on the design of antiviral RNAi constructs and their application in transgenic plants. More comprehensive reviews have previously described various types of RNA-mediated virus resistance and protein-based PDR [17–19].
Production and action of viral siRNAs in virus-infected plants
different steps of the biogenesis and/or action of viral siRNAs as well as plant miRNAs [34]. One of the common strategies evolved by RNA viruses involves the binding of sRNAs by a viral suppressor protein, preventing RISC assembly [35]. This strategy may account for large quantities of viral siRNAs accumulating in some virus-infected plants without any apparent antiviral effect. Furthermore, viruses can evade silencing by evolving the protective secondary structures that prevent RISC access and by expressing from highly structured regions the decoy dsRNAs that engage the silencing machinery in massive siRNA production and thereby protect other regions of the virus genome from repressive siRNAs [24,33,36,37].
In all virus-infected plants the RNA silencing machinery generates siRNAs from the entire virus genome sequence in both sense and antisense polarities. Indeed, complete genomes of DNA and RNA viruses and viroids can be reconstructed by siRNA sequencing and de novo assembly [20]. Nonetheless, viral sequences are targeted differentially, resulting in siRNA hotspots [20]. The hotspot profile and sequence features of viral siRNAs, which reflect relative plant defense and viral counter-defense activities, should be taken into consideration for better design of antiviral RNAi constructs.
Design of antiviral RNAi constructs
The genetic requirements for biogenesis and action of viral siRNAs have been thoroughly investigated in Arabidopsis and, to a lesser extent, in Nicotiana benthamiana and rice. All four Arabidopsis DCLs are involved in the generation of viral siRNAs [21], but their impact on antiviral defense depends on the infecting virus. RNA viruses with cytoplasmic replication/transcription are targeted mostly by DCL4 and DCL2 generating 21-nt and 22-nt siRNAs [21,22,23], while DNA viruses with nuclear replication and/or transcription are, in addition to DCL4 and DCL2, targeted by DCL3 and DCL1 generating 24-nt and 21-nt siRNAs, respectively [21,24,25]. Similarly, RDR1, RDR2 and RDR6 are involved in defense against RNA viruses and production of viral secondary siRNAs [26–28], while neither RDR1, RDR2 nor RDR6 contribute to the biogenesis of DNA virus-derived siRNAs [24,25]. Among multiple AGOs implicated in plant antiviral defense [29], AGO2 preferentially binding 21–22-nt 50 A-RNAs plays a major role, owing to its ability to bind viral siRNAs [28,30] and form RISCs with cleavage/slicer activity [31], while AGO1 binding 21–22-nt 50 U-RNAs plays both direct and indirect roles via its association with viral siRNAs [28,32] or plant miRNAs [30], respectively. AGO4 binding 24-nt 50 A-RNAs has been implicated in defense against DNA viruses, although its association with 24-nt viral siRNAs was not confirmed [24,33].
The originally designed hairpin RNAs contained a spacer between the inverted repeats [39,42]. Replacement of the spacer with an intron increased antiviral immunity [39]. Intron excision may help to align the complementary hairpin arms, promoting the formation of a duplex. Alternatively, splicing may facilitate the hairpin’s passage from the nucleus, or create a smaller, less nucleasesensitive loop. However, neither a bigger loop size nor an unpaired 50 -extention interfered with hairpin RNAmediated silencing [43]. Thus, the spicing event itself is important, likely because it is coupled with nuclear export of hairpin RNA.
To counteract these antiviral defenses, most plant viruses have evolved silencing suppressor proteins targeting www.sciencedirect.com
Concurrent with the discovery that dsRNA is a trigger of RNAi in animals [9], simultaneous expression of sense and antisense RNAs in transgenic plants was found to be a more potent inducer of antiviral silencing than separate expression of sense or antisense RNAs [38]. This finding paved a way for subsequent design of RNAi constructs expressing an intron-spliced hairpin RNA with inverted repeats of a viral sequence and their successful application against RNA and DNA viruses as well as viroids [39,40,41] (Figure 1a).
In RNAi-transgenic tomato immune to tomato yellow leaf curl virus (TYLCV, DNA begomovirus) [44], a 200-nt intron RNA accumulated at high steady state levels, while the hairpin RNA composed of 700-nt arms was fully processed into highly-abundant 21–22 nt siRNAs and less abundant 24-nt siRNAs, as demonstrated by sRNA sequencing combined with blot hybridization [45]. These findings together with previous characterization of genetic requirements for the biogenesis of intronhairpin RNA -derived siRNAs in Arabidopsis [23] suggest that the hairpin RNA transcript is efficiently spliced and transported to the cytoplasm where it is processed by DCL4 and DCL2 into 21-nt and 22-nt siRNAs, respectively, while a small proportion of hairpin RNA is retained in the nucleus and processed by DCL3 into 24-nt siRNAs (Figure 1a). Analysis of 50 -nucleotide identities of the transgenic siRNAs showed their possible association with Current Opinion in Virology 2017, 26:28–35
30 Engineering for viral resistance
Figure 1
Inverted repeat
Viral sequence
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(b) RNAi transgene
Inverted repeat
Viral sequence Term.
35S Pro
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cell-autonomus effectors
-2,000 -4,000 Current Opinion in Virology
The model for biogenesis and the single-nucleotide resolution maps of intron-hairpin RNA transgene-derived siRNAs. (a) The transgene is shown schematically with the promoter, the inverted repeat and the intervening intron boxed. Pol II generates a capped and polyadenylated transcript that folds back to form a duplex. The resulting hairpin undergoes splicing and nuclear export. A fraction of unspliced hairpin is processed by DCL3 into 24-nt siRNAs mediating phloem-mobile silencing, whereas the spiced hairpin is processed by DCL4 and DCL2 into 21-nt and 22-nt siRNAs mediating cell-autonomous silencing of the target virus. (b) Small RNAs from virus-free RNAi-transgenic tomato plants, sequenced and mapped to the transgene cassette reference sequence as describes in [45]. The transgene cassette is shown schematically with the 35S promoter, the TYLCV Rep gene inverted repeats, the intervening intron and the terminator boxed. The histograms plot the number of 21-nt, 22-nt, and 24-nt siRNA reads at each nucleotide position of the transgene expression cassette in sense (bars above the axis) and antisense (bars above the axis) orientation. Note that the mapping tool distributed identical reads equally (50–50%) between the two inverted repeat sequences, resulting in mirroring profiles of the siRNA hotspots. The read number scales are different between more abundant 21–22-nt siRNAs and less abundant 24-nt siRNAs.
multiple AGOs including the antiviral AGO1, AGO2 and AGO4 [45]. Moreover, the transgenic siRNAs accumulated at much higher levels (6–8% of total sRNAs) than TYLCV viral siRNAs in non-transgenic plants (2–3%), thus explaining their durable protective effect [44,45].
did not result in antiviral immunity [49,50]. In those cases where hairpin RNA transgenes failed to exert antiviral effects, quantities and size/50 -nucleotide/hotspot profiles of transgenic siRNAs were not analyzed by deep sequencing.
It has been reported that hairpin RNA transcription does not necessarily guarantee triggering antiviral RNAi [46]. However, a spacer-hairpin RNA investigated in that study would be inefficiently transported to the cytoplasm for DCL4-mediated and DCL2-mediated production of siRNAs targeting a cytoplasmic RNA virus [46]. The higher accumulation levels of hairpin RNA transgenederived siRNAs generally correlated with the higher resistance to both RNA and DNA viruses [42,47,48]. Nonetheless, in some cases transgenic siRNA production
Besides Fuentes et al. [45], several studies have applied deep sequencing to characterize intron-hairpin RNAderived siRNAs in transgenic plants resistant to RNA and DNA viruses [51,52,53,54–56]. Notably the hotspot profiles of transgenic siRNAs were generally quite similar to those of virus target sequence-derived viral siRNAs, and the quantities of transgenic siRNAs exceeded those of viral siRNAs in non-transgenic plants, thus supporting the notion that hairpin RNA transgenes pre-activate and boost the natural antiviral mechanism.
Current Opinion in Virology 2017, 26:28–35
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Transgenic RNAi-mediated resistance to plant viruses Pooggin 31
Leibman et al. [51] reported that the intron-hairpin RNA transgenic cucumber line with higher siRNA quantity (42% of total sRNAs) exhibited broad-spectrum resistance to both target and non-target RNA potyviruses, compared to the transgenic line with lower siRNA quantity (13%) exhibiting resistance only to the target potyvirus. A notable difference between the two lines is that the former had an increased RDR1 expression and accumulated 24-nt transgenic siRNAs, whereas the latter had no RDR1 induction and accumulated only 21–22 nt siRNAs [51], suggesting a contribution of RDR1 and/or 24-nt siRNAs to broader resistance. Zhao and Song [52] reported that RNAi-transgenic cherry plants resistant to an RNA ilarvirus accumulated siRNAs of low abundance (0.2% of total sRNAs), but the predominance of 24-nt and 21-nt classes with a strong bias to 50 A and 50 U, respectively, could explain the protectiveness of low abundance siRNAs. Moreover, the follow-up study demonstrated mobility of the 24-nt siRNAs from the transgenic cherry rootstock to a non-transgenic scion, which conferred antiviral resistance in the grafted scions [53]. The mobility of a small proportion of intron-hairpin RNA-derived siRNAs from the transgenic rootstock to a non-transgenic scion was also reported in tomato plants. However, only 21–22-nt siRNAs were found in the grafted scions, since 24-nt siRNAs were not expressed in the rootstock, and the scions were susceptible to target begomovirus infection [55]. Thus, both cellautonomous 21–22-nt siRNAs and mobile 24-nt siRNAs contribute to virus resistance in RNAi-transgenic plants (Figure 1a). This is consistent with the findings that mobile 24-nt sRNAs can direct epigenetic silencing in Arabidopsis [7,57]. The sRNA sequencing also revealed that the production of intron-hairpin RNA-derived siRNAs is restricted to the viral inverted repeats [45,54] (Figure 1b). Thus, secondary siRNA production outside of the hairpin arms (i.e. promoter, intron and terminator), which may result in transgene silencing, is inefficient: this explains robust siRNA expression and durable antiviral immunity of intron-hairpin RNA-transgenic plants for several generations under field conditions [45].
The choice of an RNAi target region within a virus genome RNAi constructs with different lengths of a hairpin arm/ viral target sequence have been successfully used in various plant species [18]. Both short (50–150 bp) and longer (up to 2.5 kbp; [58]) hairpins were protective. More diverse siRNA species derived from longer hairpins are expected to be more protective as they target longer viral sequences. However they may also cause off-target effects, that is, siRNA-directed silencing of host plant gene(s) with a negative impact on plant development or physiology. To avoid off-target effects, artificial miRNA transgenes designed to express a 21–22 nt miRNA-like www.sciencedirect.com
species cognate to a target virus have been employed [59]. However, viruses quickly evolve to mutate a short target sequence and thereby evade the artificial miRNAmediated resistance in transgenic plants [60,61]. Moreover, artificial miRNA-mediated virus resistance was shown to be non-transmissible through graft junction [62], possibly due to the lack of mobile 24-nt siRNAs. This issue is important for woody perennial plants where grafting of non-transgenic scions on transgenic rootstocks is considered as the most promising antiviral strategy [63]. The choice of target sequences within a virus genome can be important to achieve resistance against both RNA and DNA viruses. Indeed, transgenic RNAi resistance to segmented RNA viruses in rice required identification and targeting of a viral ‘Achilles’ heel’ gene [64]. The reason why some of the viral target genes/genome regions are resilient to transgenic siRNAs was not systematically investigated. It is conceivable that GC-rich target sequences would tend to form secondary structures, preventing RISC access, while AU-poor hairpin arms would generate low abundance 50 A-siRNA and 50 U-siRNA, reducing the chances to form active RISCs with AGO1, AGO2 and/or AGO4. It is also conceivable that viral genome regions that do not spawn abundant siRNAs in virus-infected plants are more optimal targets than viral siRNA hotspot regions which might deliberately produce dsRNA decoys [24,37]. Consistent with the latter hypothesis, mutations in the siRNA hotspot sequences within an RNAi target region did not break RNAi-mediated antiviral immunity [51], indicating that the transgenic siRNAs targeting non-hotspot viral sequences are sufficiently protective. In the case of DNA begomoviruses, targeting a bidirectional promoter region by intron-hairpin RNA resulted in recovery from initial virus infection in transiently transformed mungbean seedlings [40] and transgenic cassava plants [65], whereas targeting an AC1/Rep coding sequence resulted in complete antiviral immunity in transgenic cassava [48], tomato [44] and common bean [66] plants. In the latter cases, the transgenic siRNAs likely mediated both posttranscriptional silencing of the viral Rep mRNA and transcriptional silencing of the AC2 gene whose promoter elements are located in the upstream AC1 target sequence.
How to achieve broader and more durable antiviral resistance through RNAi? Because of sequence specificity of RNAi, the spectrum of virus resistance is generally restricted to viral strains or closely related viruses with greater than ca. 90% sequence identities. The broader spectrum resistance could be achieved through transgenic expression of hairpin RNAs with chimeric arms composed of sequences from two or more viruses [47,58,67,68], or cassettes composed of Current Opinion in Virology 2017, 26:28–35
32 Engineering for viral resistance
several separate intron-hairpin RNAs targeting different viruses [69]. The durability of RNAi-mediated resistance may depend on relative abilities of target viruses to overcome RNAi. Indeed, viral strains encoding stronger silencing suppressors could in some cases brake RNAi-mediated resistance [70]. Likewise, a non-target virus can potentially suppress RNAi resistance to a target virus. Furthermore, a target virus can potentially mutate the RNAi target sequence or replace it by recombination with a distantly related virus, as was recently reported for RNAi-transgenic tomato plants under open field conditions [45]: some of the asymptomatic transgenic plants contained a chimeric virus, in which a large region of the targeted monopartite begomovirus containing the 700 nt RNAi target sequence was replaced with a homologous region from a non-target bipartite begomovirus (Figure 2). The recombinant virus evaded the repressive action of transgenic siRNAs, because it shares only 62% homology with the hairpin arm sequence (with no stretch of nucleotide identity longer than 17 nts) [45].
Concluding remarks Despite numerous reports on successful application of transgenic RNAi against economically important plant
viruses, field trials and commercialization of RNAi-transgenic plants were carried out only in a few cases, likely because of negative public perception of genetically modified organisms. Recently, intron-hairpin RNA-transgenic common bean plants resistant to bean golden mosaic virus [66] have been accepted for commercial use in Brazil: these plants exhibited durable virus resistance under open field conditions as well as unaltered agronomic characteristics and nutritional value [71,72,73]. Other examples of transgenic crops approved for commercial use mostly include those in which virus resistance was mediated by expression of viral coat protein genes [19]. One of the concerns is that constitutive siRNA expression (under control of CaMV 35S promoter used in most RNAi transgenes) would lead to off-target effects. Indeed, the transcriptome profiling of two RNAi-transgenic tomato lines with different selection markers revealed a common set of differentially expressed genes [45]. It is not clear, however, whether the transgenic siRNAs had direct or indirect effects. Interestingly, the co-regulated genes did not include any silencing-related genes, suggesting the plant silencing machinery was not affected or saturated by the production of abundant intron-hairpin RNA-derived siRNAs [45].
Figure 2
Parental monopartite target begomovirus
Recombinant monopartite begomovirus V2
C4
V2
AC4
RNAi
RNAi C1
AC1
TYLCV
TLV 2.7 kb
2.7 kb
V1 (CP)
100% identity C2
V1 (CP)
62% identity AC2
C3
AC3
AC4 BC1
RNAi AC1
62% identity
DNA-A
AC2
DNA-B AV1 (CP)
BV1
AC3
Parental bipartite non-target begomovirus Current Opinion in Virology
Recombination-dependent evasion of antiviral RNAi in transgenic tomato plants resistant to tomato leaf curl virus (TYLCV) under open field conditions. The intron-hairpin RNA-transgenic plants were immune to TYLCV disease transmitted to all control tomato plants by viruliferous whiteflies from neighboring virus-infected plants. Molecular analysis revealed that some of the asymptomatic transgenic lines contained a recombinant virus in which a large region of the TYLCV genome (AC1/AC4/AC2/AC3 genes) was replaced with the corresponding region from an unknown virus distantly-related to two known bipartite begomoviruses. The recombinant virus, named Tomato latent virus (TLV), evaded transgenic RNAi, because an RNAi target region between AC4 and AC2 genes was only 62% identical to that of TYLCV (for details, see [45]). Current Opinion in Virology 2017, 26:28–35
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Transgenic RNAi-mediated resistance to plant viruses Pooggin 33
As an alternative to transgenic RNAi, non-transgenic delivery of dsRNA or siRNAs to plant tissues has been considered, since topical dsRNA application was shown to confer resistance against plant viruses [74]. This approach is still hampered by transient protective effects and high costs of dsRNA synthesis, although cost-effective methods of dsRNA production have been developed [75] and delivery methods are being improved to stabilize dsRNA and thereby prolong the antiviral RNAi effect [76].
14. Zvereva AS, Pooggin MM: Silencing and innate immunity in plant defense against viral and non-viral pathogens. Viruses 2012, 4:2578-2597.
Conflict of interest
19. Khalid A, Zhang Q, Yasir M, Li F: Small RNA based genetic engineering for plant viral resistance: application in crop protection. Front Microbiol 2017, 8:43.
The author declares no conflict of interest.
Acknowledgements I thank Alejandro Fuentes for fruitful collaboration on RNAi-transgenic tomato plants. The work was supported by Swiss National Science Foundation [grant N 155737].
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