When an Intruder Comes Home: GM and GE Strategies to Combat Virus Infection in Plants

Abstract

Viruses are silent enemies that intrude and take control of the plant cell’s machinery for their own multiplication. Infection by viruses and the resulting damage is still a major challenge in the agriculture sector. Plants have the capability to fight back, but the ability of viruses to mutate at a fast rate helps them to evade the host’s response. Therefore, classical approaches for introgressing resistance genes by breeding have obtained limited success in counteracting the virus menace. Genetic modification (GM)-based strategies have been successful in engineering artificial resistance in plants. Several different approaches based on pathogen-derived resistance, antisense constructs, hairpin RNAs, double-stranded RNA, etc., have been used to enhance plants’ resistance to viruses. Recently, genome editing (GE) strategies mainly involving the CRISPR/Cas-mediated modifications are being used for virus control. In this review, we discuss the developments and advancements in GM- and GE-based methods for tackling viral infection in plants.

1. Introduction

Viruses constitute nearly half of the microbial pathogens that are accountable for the emerging and re-emerging epidemics worldwide [1]. They also pose serious challenges to agriculture by causing major plant diseases like the bunchy top of bananas, tobacco mosaic, wheat streak mosaic, potato tuber necrotic ring spot, cucumber green mottle mosaic, tomato yellow leaf curl, African cassava mosaic, and so on. So far, 131 families containing 803 genera and 4853 species of plant virus are reported by International Committee on Taxonomy of Viruses [2]. Viruses are responsible for a worldwide crop loss of $30 billion annually and is predicted to reach up to $60–80 billion [3,4,5]. Plant viral diseases threaten global food security as they can cause severe food limitations for the world population, which is projected to elevate from 8 billion in 2022 to 10 billion by 2050 [6,7].

For the resolution of any problem, it is important to implement appropriate prevention and control measures. However, viruses cannot be controlled by chemicals like pesticides, although their carriers or vectors can be restrained to some extent [8]. This simply leaves the possibility of finding or generating plant species with inherent resistances to viral infections. A silver lining emerged from McKinney’s observation in 1929 that the mild infection caused by the Tobacco mosaic virus (TMV) green mosaic strain protected the tobacco plants from severe infection by the TMV yellow mosaic strain, which suggested the existence of mechanisms for cross-protection [9]. This phenomenon involved the defense response of the plant to a mild virus and the formation of a memory of the response in the host plant to combat the challenge posed by a related virus [10]. These early observations prompted the search for host-based resistance genes and an understanding of plant–virus interactions [11,12].

Studies performed by different groups over the last century have shown that plants have several physical barriers like cuticles, thorns, trichomes, etc., and chemical barriers like the accumulation of lignin, callose, terpenoids, phenolics, etc., for primary protection against viral invasion [13]. In addition, they have several complex and overlapping natural defense mechanisms to tackle the viruses that manage to invade. Plant immunity can be categorized as a pathogen-triggered immunity (PTI) and an effector-triggered immunity (ETI). A PTI relies on the effective detection of the invading pathogen and limits their growth and spread across host cells [14]. For successful invasion, the pathogens produce different types of avirulence factors (AVRs) for evading or suppressing PTI pathways. The host cells, in turn, produce specific resistance (R) proteins that can recognize the effectors [15]. This R–AVR interaction leads to the activation of an ETI [16,17]. The first line of defense in cases of viral pathogens is the activation of the RNA silencing or RNA interference (RNAi) pathways by viral dsRNAs. The viral triggers for PTI and ETI are not clearly defined, though the viral dsRNAs are generally paralleled to the PAMPs. Some virus-encoded proteins have the ability to suppress the silencing process and they can act as effectors that lead to the generation of an ETI.

Different R genes such as NBS-LRR, RTM1, RTM2, and RTM3 were discovered in Arabidopsis thaliana, which provided resistance against viruses, especially the potyviruses, by preventing the systemic spread through the phloem [18,19]. In addition, the mutations or recessive alleles of certain host-encoded factors which are essential for virus replication provided a unique recessive resistance against plant viruses [20].

The selection of dominant and recessive genes associated with virus resistance played an important role in the breeding of naturally resistant plant varieties as a cheaper and better alternative for controlling viral diseases [5]. Classical approaches such as the introgression of resistance genes by marker-assisted selection (MAS), quantitative-trait loci (QTL) mapping and gene pyramiding were extensively used to tackle the menace of viruses. Another reverse genetics approach known as TILLING (Targeting induced local lesions in genomes) was used to create novel mutations in alleles in order to repress the expression of essential factors that are needed by a virus to complete its infection cycle [21]. However, there were several bottlenecks, such as the non-availability of markers near the R genes, the transfer of undesired traits, and the screening of plants for several generations, which made the process tedious and time-consuming, thereby limiting the success of these techniques.

The era of genetic engineering accelerated the process by allowing the incorporation of genes in the plant genome as per the requirement. In the early 1980s, the concept of pathogen-derived resistance (PDR) laid the foundation of artificial resistance. PDR employed the use of virus proteins like the coat protein (CP) to disrupt virus replication by inactivating the viral components [11]. Subsequent studies unraveled the role of RNA silencing in symptom recovery and cross-protection [22,23]. It also strengthened the possibilities for employing RNA silencing for generating virus resistance in plants. However, the silencing pathways have some limitations due to the presence of silencing suppression activity in the viral-encoded proteins. In recent times, CRISPR/Cas-based tools are being used to edit the plant genome for generating virus-resistant plants.

Both genetic modification (GM) and genome editing (GE) approaches are quite efficient in accelerating the generation of virus-resistant plant varieties without the need of backcrossing. These techniques are very precise and time-saving as compared to traditional breeding. In particular, for virus management, many transgenic plants, such as the squash, papaya, bean, and cassava, have received approval for field trials and commercial cultivation in various countries [24]. The various aspects and examples are described in the following sections.

2. RNA Silencing as a Major Antiviral Weapon

The discovery of non-coding small RNAs and their role in gene silencing provided a paradigm shift in the understanding of a host’s defense against a virus and the generation of virus resistance. RNA silencing is an evolutionarily conserved, fundamental cellular mechanism for regulating gene expression [25]. The process involves small RNA mediated degradation or the inactivation of specific mRNAs in a sequence-dependent manner [26]. In plant immunity, it contributes as the first line of defenses against virus infection, as is often referred to as virus-induced gene silencing (VIGS). Earlier, it was understood that the genomes of RNA viruses and viroids were subject to RNA silencing [27,28], but later it was also shown to be effective against DNA viruses such as Geminiviruses, Caulimoviruses and Tungroviruses (which have dsDNA with RNA intermediate) [29,30].

The RNA-silencing machinery is triggered by the production of double-stranded (ds) RNA either as a replicative intermediate or a self-folded complementary region of single strand (ss) viral transcript (Figure 1). The dsRNA is diced by the microprocessor protein complex containing dicer-like proteins (DCLs) to generate 20–24 nt small RNAs [31]. The small RNAs derived from the virus are normally categorized as viral small interfering RNAs (vsiRNAs). They are loaded onto an RNA-induced silencing complex (RISC) to direct transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS) [31,32]. In TGS, the target gene gets methylated to block transcription [33,34], whereas in PTGS, the target mRNA is cleaved to block translation [35]. The vsiRNAs also act as mobile signals as they can spread from cell to cell [36,37]. These primary siRNAs were also amplified with the help of SGS3 and RNA dependent RNA polymerase (RdRP), RDR6, to produce secondary siRNAs to provide protection throughout the plant [31,38].

2.1. Role of Transcriptional Gene Silencing (TGS) in Viral Infection

RNA silencing has a great impact on regulating viral genes as well as host genomes via epigenetic modifications. The small RNAs initiate the RNA-directed DNA methylation (RdDM) of complementary DNA sequences [39,40], which reduces or blocks the transcription of mRNA, so the gene undergoes silencing [41,42]. Some proteins like AGO4, DCL3, RNA polymerase IV, RDRs, which are involved in TGS could also play a role in systemic silencing. Many small RNAs as well as dsRNAs were reported to have a role in targeted methylation by triggering RdDM.

Studies have shown that a typical small RNA-mediated methylation pathway involves transcription from the target locus by RNA polymerase IV [43]. The ssRNA is converted into dsRNA through the action of RdRP/RDR2, which is then diced by DCL3. The siRNAs thus generated associate with AGO4 containing RISC and polymerase IV complexes to search for the target DNA sequences. At the target site, RNA polymerase V is recruited to form an AGO4:siRNA:PolV complex which attracts de novo methyltransferases [44]. Methyltransferases, like Domain rearranged methyltransferase (DRM) and Kryptonite 2 (KYP2), are recruited at the target site along with the Defective in RNA-directed DNA methylation 1 (DRD1), Defective in meristem silencing 3 (DMS3), and Deficient in DNA methylation 1 (DDM1) [45]. Cytosine methylation engages the histone deacetylases and other methyltransferases to initiate histone modifications [46]. It has been shown that methylation in the promoter region, rather than the coding region, is more effective for silencing [47]. Moreover, Chromomethylase 2/3 (CMT2/3) is involved in CHG/CHH methylation while Methyltransferase 1 (MET1) has a role in CG methylation. CG and CHG can be maintained after cell division and without RNA triggers, but CHH methylation cannot [39,48,49].

DNA viruses can be easily targeted through TGS, because of their ssDNA and the dsDNA replication process (Figure 1). The replication intermediates of Pararetroviruses and Geminiviruses form mini chromosomes, which are common targets of TGS [50,51,52]. VIGS was shown to initiate RdDM and methylation-deficient mutants of Arabidopsis were hypersusceptible to infection [45]. The accumulation of vsiRNAs to threshold levels at the target DNA recruits DRM2 for initiating de novo methylation [53]. The methionine cycle (MTC) plays a crucial role in small RNA-mediated methylation during viral infection by maintaining a continuous supply of S-adenosyl methionine (SAM) and the removal of the methyl transferase inhibitor, S-adenosyl-homocysteine (SAH). S-adenosyl-L-homocysteine hydrolase (SAHH) converts SAH to L-homocysteine (HCY) and then HCY is converted into methionine by Methionine synthase (MS). SAH has been shown to inhibit HUA enhancer 1 (HEN1), which is responsible for siRNA stability through the 2′-O-methylation of their 3′ pentose sugar. It was hypothesized that viral-encoded RNA silencing suppressor proteins like HC-Pro interact with SAM synthase and reduce its activity which results in decreased levels of SAM [54]. Mixed virus infections of potyvirus and potexvirus create imbalances in MTC and GSH biosynthesis which induce an oxidative burst and lead to more symptoms in infected plants [55].

2.2. Role of Post Transcriptional Gene Silencing (PTGS) in Viral Infection

The replicative intermediates of RNA viruses and the transcripts of DNA viruses are targets of PTGS in the cytoplasm [52]. Plants show symptoms of recovery when the vsiRNAs are generated and spread through the vascular system. Arabidopsis mutants of sgs3, ago1, sgs2 showed greater susceptibility to viral infection because their silencing pathways were compromised [56]. Studies with Tomato bushy stunt virus (TBSV) infection in Nicotiana benthamiana and the Potato virus X (PVX) and Turnip crinkle virus (TCV) in Arabidopsis have shown that the antiviral defense involves the AGO2 pathway [57,58,59].

To counteract RNA silencing, viral proteins contain a suppressor of RNA silencing activity (Figure 1). The VSRs interact with the components of PTGS at multiple points. They can interrupt the silencing process by making dsRNAs unavailable for DCL action, inhibiting the siRNAs’ transport, quenching the siRNAs to prevent their loading in RISC and the inactivation of RISC [60]. For instance, the 2b protein, the VSR of the Tomato aspermy virus (TAV), shows non-specific binding with siRNA and dsRNA [61]. Whereas V2, the VSR is of the Tomato yellow leaf curl virus, binds with SGS3 which has an important role in dsRNA amplification [62,63].

3. GM Approaches

These approaches involve the over-expression or knock-down of genes using a variety of genetic engineering tools. The methodology usually involves cloning the gene of interest (trans-gene) into the desired vector in either a sense or antisense orientation followed by its transformation in plants [64]. Many old reports are available on engineering virus resistance in plants via the transgenic approach [65,66].

3.1. Pathogen-Derived Resistance

This approach involves the use of the pathogen’s own genetic material to generate a resistance in the host plant. The concept of PDR was first presented by Sanford and Johnston in 1985 [11] and a year later it was proven to be successful in cases of viral infections. The transgenic plants expressing the coat protein (CP) gene of TMV were shown to exhibit a resistance against infection [67]. This ground-breaking discovery initiated the development of various virus-resistant transgenic plants [6]. PDR can be classified into different types depending on the strategy used to generate the resistance towards the virus in the host plant.

3.1.1. Protein-Mediated Resistance

This process entails the expression of viral-encoded proteins like CP, MP (movement protein) or Rep (replicase) in plants to counteract viruses [6]. In 1986, Beachy’s group first demonstrated that the expression of the CP gene of TMV in transgenic tobacco plants could provide considerable protection against viral infection [68]. It was observed that the expression of these viral proteins interfered with the disassembly of the virus by the formation of pseudo-capsids [6]. Several transgenics were made using the CP of the Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV), PVX, Plum pox virus (PPV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Tomato yellow leaf curl virus (TYLCV), Tomato mosaic virus (ToMV), and so on [69].

PDR also provided cross-protection against infection by related viruses. This technique was more effective in cases of closely related viruses than those which are distantly related [70]. When TMV CP-transgenic plants were infected by different viruses of the Tobamovirus family, their inhibition was differentially compromised. Viruses like the ToMV, Pepper mild mosaic virus (PMMV) and Tomato mild green mosaic virus (TMGMV) were inhibited completely while no resistance was observed against others like CMV, African mosaic virus (AMV), PVX, and Potato virus Y (PVY), though there was a decrease in symptoms and spread [71]. In field trials also, CP-TMV expressing tomato and CP-PVX/PVY expressing potato transgenics performed better than the wild-type [72].

In cases of PVX infection, it was shown that the interaction of the 5′ end of viral RNA with CP expressed in the host plant suppressed the expression of the RdRP encoded by the virus [73,74]. The region and quantity of protein expression are important determinants for the generation of resistance. For instance, if CP is expressed in mesophyll but the viral RNA is introduced in epidermal cells then resistance did not develop [75]. The expression of mutant CP in transgenics also resulted in a resistance against wild-type CP, as the mutant CP could interact with the wild-type CP subunits or the host factors, which were involved in virion disassembly [76,77]. The extent of the aggregation of the CP correlated with the level of the coat protein-mediated resistance (CPMR) [78]. This observation suggested that homology between the amino acid sequences of the challenging viruses provided cross-protection in the transgenic organism [6].

Likewise, the MPs, which have a role in the cell-to-cell spread of the virus, were also utilized for triggering PDR. This technique worked better for large genera of viruses irrespective of the viral MP expressed in plants. The expression of defective or mutant MPs could also block virus transmission since the mutant MPs competed with the wild-type MPs of the virus for the plasmodesmatal sites [79]. Mutant TMV-MP provided resistance against Potexviruses, Tobamoviruses, and Cucumoviruses [80]. Several groups tried to multiplex this approach by generating transgenics with more than one type of MP by using overlapping regions of the ORFs which are also known as a triple gene block (TGB). However, this approach did not provide broader resistance, possibly due to the insufficient interaction of TGB with the plasmodesmata [81].

In subsequent studies, it was shown that the use of modified non-structural proteins like Rep/RdRP were able to provide better results even at low levels of expression [82]. Mutations in the ori or NTP-binding sites of the Rep gene (AC1) provided resistance against geminiviruses [83,84]. The Rep/RdRP proteins could directly interfere with virus replication or bind with host factors involved in the viral replication process [85]. Detailed investigations into the molecular mechanisms behind the responses demonstrated that these proteins mediated resistance mainly through the RNA-silencing machinery instead of direct protein interactions [86,87].

The commercially produced squash, ZW20, that is resistant to WMV and ZYMV became the first GM crop to receive approvals in the USA [88]. Subsequently, several other virus-resistant crops were produced. Despite several examples of the successful implementation of this technology, it faced some challenges due to evolutionary pressure provided by the expression of the transgene. The genomic content of the virus interacting with the transgenics was altered due to the recombination between the transgenic and viral CP transcripts [89]. This trans-capsidation of the CP in transgenics imparted a new potential to the infecting virus isolate. Mixed infection by two unrelated viruses was another issue, as it led to the antagonism and synergism of defense responses. For instance, different outcomes were produced during mixed infection with PRSV and Papaya mosaic virus (PapMV). In the case when plants were first infected with PRSV followed by PapMV or simultaneously inoculated with both, a synergistic response was observed. However, when the first inoculation of PapMV was followed by PRSV, an antagonistic response was observed, which caused a reduction in PRSV titers due to the activation of defense responses [90].

3.1.2. Defective Interfering Virus-Mediated Resistance

Plant viruses are often associated with sub-genomic/sub-viral RNAs known variably as satellite (sat) or defective interfering (DI) viruses [91]. The DI viruses lack a critical portion of the virus genome, which makes them defective for replication. They require the presence of the complete functional virus, known as the helper virus, for their replication and encapsidation [92]. DI viruses are basically the defective versions of helper viruses formed by mainly deletions, duplication, and inversion processes [93]. There is often a synergistic interaction between the DI virus and its helper as they can produce mild symptoms on one host plant but elicit severe symptoms on another host or in combination with a different strain of a helper virus [94,95]. According to some reports, a DI virus can attenuate symptoms of an unrelated helper virus by inhibiting their multiplication and long-distance transport [96,97]. Hence, they were used in transgenic approaches to decrease virus symptoms and/or titers [92]. As for satellite RNAs, they can compete with the helper virus genome for binding to replicase for replication, which indirectly suppresses virus replication [97]. In 2015, it was shown that CMV sat-Y-derived siRNAs bind to the viral encoded suppressors of RNA silencing (VSR), 2b of CMV and P19 of Tombusvirus, causing the release of the VSR-bound helper viral siRNAs resulting in the down-regulation of the viral target genes [98]. The major limitation of this strategy was a probable risk that the DI virus and satellites could mutate to a virulent form in conjunction with the infecting virus in the transgenic plants.

3.2. Non-Coding RNA Shields in Virus Resistance

RNA silencing has been effectively used for generating virus resistant plants [99]. A variety of small RNAs have been used through various strategies to trigger the RNA silencing-mediated defense responses against viruses (

Table 1. List of small RNA-based GM approaches used against plant viruses.

Strategy	Virus	Target	Plant	References
hpRNA (inverted repeat of target gene which forms a loop structure for transcriptional silencing	Tobacco mosaic virus	Movement protein	Tobacco	[102]
	Cucumber mosaic virus	Replicase		[103]
	Plum pox virus	P1 and HC-Pro		[104]
	Cucumber green mottle mosaic virus	Coat protein
	Potato spindle tuber viroid	Viroid sequence	Potato	[27]
hpRNA (single RNA molecule that folds due to self-complementarity to form hairpin structure)	Tomato yellow leaf curl virus	Replicase (C1) Coat protein	Tomato	[105,106]
	Potato virus X	ORF2	Potato	[107]
	Potato virus Y	HC-Pro
	Potato leafroll virus	CP
	Papaya ringspot virus	CP	Melon	[108]
	Soybean dwarf virus	CP	Soyabean	[109]
	Soybean mosaic virus	HC-Pro (VSR)		[110]
	White clover mosaic virus	Replicase	White clover	[111]
	African cassava mosaic virus	Rep (AC1)	Cassava	[112]
	Banana bunchy top virus	Replicase	Banana	[113]
	Citrus tristeza virus	P20, p23, p25 (VSRs)	Mexican lime	[114]
DI Virus-derived RNA (interfere during virus replication)	Potato virus Y Tomato busgy stunt virus Grapevine fanleaf virus	Coat protein Movement protein	Tobacco	[115]
dsRNA (two complementary RNAs base-paired together)	African cassava mosaic virus	DNA-A and DNA-B	Tobacco	[116]
	Rice grassy stunt virus	pC5, pC6	Rice	[117]
	Rice tungro bacilliform virus	ORF IV	Rice	[28]
amiRNA/single monocistronic
AthMIR156 backbone	Cucumber green mottle mosaic virus	CP	Tobacco	[118]
AthMIR167b backbone	Potato virus X	P25	Tobacco	[119]
	Potato virus Y	HC-Pro	Tobacco
amiRNA/single polycistronic
OsaMIR395 backbone	Wheat streak mosaic virus	5′UTR + P1 + HC-Pro + P3	Wheat	[100]
amiRNA/single monocistronic in tandem repeats
AthMIR159a backbone	Potato virus X,	P25	Tobacco	[119]
	Potato virus Y	HC-Pro
	Turnip mosaic virus,	HC-Pro
	Turnip yellow mosaic virus	P69 (TYMV)
amiRNA/multiple monocistronic in trans
AthMIR159a backbone	Turnip mosaic virus	CP + HC-Pro	Arabidopsis	[120]
AthMIR390a backbone	Tomato Bushy stunt virus	5′ terminal (+) RNA	Tobacco	[121]
ta-siRNAs AthTAS3a backbone	Turnip mosaic virus, Cucumber mosaic virus	Multiple genomic positions	Arabidopsis	[122]
syn-tasiRNA/single polycistronic
AthTAS1c backbone	Tomato spotted wilt virus	RdRP	Tobacco	[123]

). The regions of the viral genome that encode VSRs, e.g., P69, HC-Pro, and AC2, are usually selected as the targets of the small RNAs. The expression of small RNAs can be from various precursors, which may be in cis or trans and single or polycistronic [100,101]. Initial GM approaches involved the use of antisense constructs or hp-RNAs as precursors to generate a pool of siRNAs, which could target the viral transcripts. Subsequently, these were replaced by more specific tools through the use of constructs expressing antiviral artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs).

3.2.1. microRNA Shield

MicroRNAs (miRNAs) are genome-encoded small RNAs that are processed by DCL1 from imperfectly paired, hairpin precursor RNAs [124,125]. The miRNAs belong to large gene families and each miRNA can modulate a plethora of targets involved in plant immunity and phytohormone pathways [126]. High throughput sequencing tools have provided useful insights for identifying the miRNAs involved in virus resistance. miR156, miR395, miR159, miR166, miR168, miR160, and miR444 have important roles in virus attacks [127,128,129,130]. A recent report has shown that a novel miRNA, Seq119, is down-regulated during Rice stripe virus (RSV) infection. The overexpression of this miRNA reverses the symptoms caused by RSV [131].

In many cases, amiRNAs have been used to over-express a synthetic sequence designed to target a key viral gene. In Arabidopsis plants, amiRNAs were used first to target TYMV and Turnip mosaic virus (TuMV) [132]. This technique has the advantage of enhanced specificity and reduced off-target effects. The amiRNA is composed of two components, namely, a miRNA precursor (pre-miRNA) scaffold and a synthetic RNA insert. Special consideration is placed on the selection of the pre-miRNA backbone to ensure that the correct amiRNA is processed by the cellular machinery [133]. Studies with TuMV population dynamics showed that inadequate levels of amiRNAs build up more mutations in the target site and reduce the efficacy of virus resistance [134].

To synthesize the amiRNA, the miRNA/miRNA* sequence of the pre-miRNA is replaced by a synthetic sequence complementary to the viral target by performing an overlapping PCR [135]. The sequence of the amiRNA is designed to have maximum complementarity with the target and minimum hybridization energy while ensuring sufficient mismatches to retain the secondary structures or bulges [136,137]. Instability at the 5′ end and the avoidance of mismatches at the 10 or 11th nucleotides are also crucial parameters. The design of an effective amiRNA also necessitates the incorporation of Uracil (U) at the first base and Adenine (A) or U at the 10th nucleotide position for making amiRNA biologically active [136,138]. Targeting an exposed region in a viral genome can increase the chances of DCL accessibility and the efficiency of the amiRNA [139]. Tomato transgenics overproducing the amiRNAs to silence the conserved regions of ToLCV- AV2/AC2 genes could tolerate various leaf curl viruses of tomatoes [140]. The amiRNA technique faced the limitations of its stringent design principles and the level of amiRNA expression.

3.2.2. siRNA Shield

The siRNAs are 21–24 nt in length and are derived from perfectly paired dsRNA molecules [141,142]. The siRNA-mediated gene silencing serves as a primary defense mechanism against plant viruses [52,143,144,145,146]. The transitivity of siRNAs is useful in propagating the silencing signal through the production of secondary siRNAs [31,147].

It was observed that better virus resistance was achieved when a dsRNA of the viral sequence was expressed in a plant cell compared to the case of expressing either sense or antisense viral RNA [148]. This resulted in the construction of intron splice-able hairpin RNA (hpRNA) constructs with viral gene or gene fragments. The hpRNA constructs offered the strongest resistance to RNA and DNA viruses [149,150,151]. The GM approaches involve the expression of short-hairpin RNA (shRNA) constructs as precursors of the siRNAs in the plants to tackle the virus. The constructs are mobilized into the Agrobacterium and stably integrated into plants [152]. In the majority of cases, the VSR genes have been used as a silencing target to generate virus resistance [150]. In Brazil, a transgenic bean resistant to a Bean golden mosaic virus (BGMV) was created by expressing a hpRNA of Rep gene, and it is being commercially cultivated [149].

Tomato plants expressing hpRNA corresponding to the TYLCV-Rep coding sequence produce 21 and 22 nt siRNAs and are resistant to TYLCV [153]. Similarly, Cassava transgenics containing hpRNA homologous to the overlapping region of South African cassava mosaic virus (SACMV)-Rep and the AC1/AC4 were tolerant to cassava mosaic disease [154]. In another study, three distinct intron hpRNA constructs comprising sequences of AC2, AC4, and a fusion of AC2 and AC4 (AC2 + AC4) of seven begomoviruses were used. All transgenic lines showed a resistance against MYMIV infection when compared with untransformed controls [155].

3.2.3. Synthetic tasiRNA Shield

The tasiRNAs are derived from non-protein-coding TAS transcripts that are capped as well as poly-adenylated and contain a binding site mostly for 22 nt miRNAs [156]. The miRNA cleavage products are stabilized by the Suppressor of Gene Silencing 3 (SGS3) and converted into a dsRNA form by RDR6 [157,158]. The dsRNA intermediate is then processed by DCL4 and dedicated dsRNA-Binding Protein 4 (DRB4) to phased 21 nt siRNAs in a ‘head-to-tail’ phased pattern. The transitive siRNAs are incorporated into AGO-RISC for targeting complementary sequences [157,158,159,160]. In Arabidopsis thaliana, eight tasiRNA-producing loci have been identified that fall into four TAS groups (TAS1–TAS4).

Since TAS precursors are able to generate multiple secondary siRNAs once they are cleaved by (with) the miRNA, they work more efficiently against the rapidly evolving viruses that can evade the single amiRNA approach. The Arabidopsis TAS DNA sequences can be engineered to silence targeted viral sequences by replacing some of the phased tasiRNA producing sequences by single or multiple siRNAs of different sequences but of an equivalent length of base-pairs. When the replacing siRNAs are processed in the tasiRNA pathway from the engineered vectors as desired, these are called synthetic-tasiRNAs (syn-tasiRNAs), which consequently silence their targets in a usual manner using the RNA-silencing machinery of the host plants. It was observed that most plants expressing higher levels of syn-tasiRNA were resistant to TSWV (Tomato Spotted Wilt Virus), while plants with particularly low levels of syn-tasiRNAs were infected. Several automated design tools have been used for designing syn-tasiRNA constructs, which are able to target distinct locations within a single RNA or multiple viral RNAs. A more efficient, long-lasting, and widespread resistance could be produced by the simultaneous co-expression of multiple syn-tasiRNAs [161,162].

3.2.4. Long Non-Coding RNA Shield

Long non-coding RNAs (lncRNAs) are 200 nt or longer and are mainly produced by the action of polymerase II [163]. The lncRNAs resemble the mRNAs in having a 5′m7G cap and 3′poly(A) tail [164,165], though their sequences show less conservation [166]. On the basis of their origin, they can be categorized as intergenic, intronic, or exonic. They can act either in cis or trans and may have sense or antisense orientations [167,168]. NGS analysis has played an important role in the identification of differentially expressed lncRNAs and their roles in host interaction studies [169]. An RNA-seq of TYLCV-resistant tomato cultivar showed that some lncRNAs such as slylnc0048, slylnc00449, slylnc0483, slylnc0531, and slylnc0934 were up-regulated while slylnc0475, slylnc0476, slylnc0673, and slylnc1052 were down-regulated after virus infection [170].

LncRNAs can act as sources for the generation of small RNAs and regulate DNA or histone methylation by guiding the DNA methyltransferase. LMT1 encoded by the Citrus tristeza virus (CTV) plays an important role in the susceptibility of host plants. The mutation in LMT1 caused an increase in the SA levels and enhanced virus resistance [171]. Recent reports have shown a relation between RNA silencing and viroid replication. Viroids are free RNA molecules which do not code for any protein and are similar to circular lncRNAs [172]. Several horticulture crops such as the apple, avocado, grapevine, peach, tomato, etc., are facing viroid infections. The Potato spindle tuber viroid (PSTVd) is a good model for studying viroid–host interactions. PSTVd-induced small RNAs cause the cleavage of several host transcripts coding for pyrophosphatase, callose, etc. [173,174].

The lncRNAs can also quench the function of small RNAs and act as target mimics of the miRNAs [167,175]. The SlLNR1 (lncRNA of tomato), involved in regulating leaf growth and development, contained sequences complementary to the vsiRNA derived from the intergenic region (IR) of the virus. In susceptible lines, viral intergenic siRNAs targeted the SlLNR1 resulting in stunted growth and the curling of leaves [176].

4. Genome Editing (GE) Approaches

Genome editing (GE) offers the most precise tool to modify a target gene by using the endonucleases to create breaks in the DNA strands that lead to both target-specific mutagenesis (addition, removal, or alteration) and gene replacement [177,178]. It offers the advantage of being less expensive, simple to design and execute, and more acceptable. Several GE techniques have been implemented against different viruses and have produced better outcomes (Table 2). By complementing it with NGS, it will become easier to identify the fast-mutating viral sequences and redesign the targets which influence the susceptibility response in host–virus interactions [179]. Genome editing is now emerging and expanding as a modern tool in biotechnology. From basic techniques such as Meganucleases (MNs), ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) to advanced techniques and the CRISPR/Cas system. GE methods rely on the introduction of specific mutations to inactivate certain target genes. Modifications like CRISPR ON and OFF, and the use of base editors and prime editors restrict the possibilities of accidental host targets.

4.1. Different Genome Editing (GE) Tools

The onset of GE lies in the use of specialized restriction endonucleases known as Meganucleases (MNs), which could target broad recognition sites of around 12–40 bp [180,181]. These nucleases create the double-stranded breaks (DSBs) that were repaired by non-homologous end joining (NHEJ), resulting in gene mutations [182]. MNs have been studied in model plant systems such as Arabidopsis, Gossypium, and maize. They exhibited a high efficiency in target recognition and a moderate mutation rate. The length of the target sequence recognized by the MN was around 68–88 bp, which was quite long as compared to others, and it did not require another endonuclease [183]. However, the major limitation was that the production of these enzymes was costlier and time-consuming. It was not easy to manipulate the MNs as their catalytic and DNA-binding domains were intermingled and became difficult to detach [184].

Zinc-finger nucleases (ZFN) emerged as specialized cutters that could cleave dsDNA sequences [185,186,187]. ZFN-induced double-strand breaks were subject to cellular DNA repair processes. ZFNs were engineered by fusing the non-specific DNA cleavage domain of Fok1 (Flavobacterium okeanokoites 1) with the Cys2-His2 ZF DNA-binding domain [188,189]. Fok1 is a type II endonuclease that can bind to DNA regions in a non-specific manner, but it can cleave the double strand only when two monomers bind to the target sequence (overall 9–18 bp). Each monomer binds to 5–6 bp-long sequences within the target region, so 4 monomers were required for generating 2 cleavage sites [190]. The breaks are repaired by homology directed repair (HDR) or NHEJ (when homologous sequences are not found) resulting in errors [191]. NHEJ creates deletions or insertions at target sites that further leads to a frame shift and results in gene knockout, while HDJ can insert new sequences at the break site that result in knock-in [192].

ZFNs were used to mutate the AC1 gene, which encodes the Rep protein, in TYLCV [193]. Artificial ZFN proteins (AZPs) have been designed using conserved sequences for blocking the DNA binding sites that are used by viruses [194,195]. For instance, AZPs bind to the IR region of the Beet severe curly top virus (BSCTV) and interfere with the binding of Rep, thus suppressing the replication of the virus in the host [196]. The ZFN-based technique allowed for easy manipulation and could target any site, but the efficiency was limited by chances of off-target cleavage. ZFN-mediated gene editing has been used in many crops such as Arabidopsis, tobacco, maize, and soybean, etc. [182,188,197,198].

TALENs (Transcription Activator-like effectors Nucleases) were produced by fusing the DNA binding domains of transcription activator-like (TAL) effectors to the DNA cleavage domains of Fok1 to induce dsDNA breaks that could be repaired by NHEJ or HDR repair mechanisms [199]. TAL effectors are produced by bacteria Xanthomonas in plants through the type III secretion system [200]. These effectors alter the transcripts of the host by binding with the promoter region via repetitive amino acid residues in the central domain and activating gene expression [201,202]. TALENs were used to generate disease-resistant plants by mutagenizing the susceptible genes in host plants [203]. TALENs were used to develop the resistance of tobacco plants against the Tobacco curly shoot virus (TbCSV) and Tomato leaf curl China virus (TYLCCNV) [204].

Recently, CRISPR-Cas has become popular in the scientific community as it has tremendous potential to improve crop traits (Section 3.2). The attractive features of this technology include its ease, efficiency, and rapidity of genetic manipulations. The CRISPR system is rapidly becoming diverse and now includes different types of base editors, prime editors, and an endogenous tRNA structure. These modifications have given it greater specificity and multiplex targeting abilities, which ultimately make GE more precise (Table 2).

4.2. Manipulation of the Genome through the CRISPR/Cas System

The CRISPR-Cas system was first identified as a unique strategy used by bacteria and archaea for adaptive immune response against invading phages or plasmids [205,206,207]. In this system, the prokaryotes were able to acquire small pieces of the infecting virus and then use it as memory for protection against further attacks. CRISPR stands for cluster regularly interspaced short palindromic repeats, and the Cas protein is the RNA-dependent DNA endonuclease. The Cas protein interacts with two special non-coding RNAs namely CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). The crRNA functions as a short guide as it consists of a unique 20-base sequence that enables target recognition. The tracrRNA is a trans-encoded small RNA, which provides the stem loop structure [208]. The fusion of the crRNA and tracrRNA generates a synthetic RNA of around 100 nucleotides (nt), called the single guide RNA (gRNA).

In the initial step, Cas9 protein binds to gRNA to form a catalytically active Cas9-gRNA duplex [209,210]. The Cas 9 protein contains two REC domains, an arginine-rich Bridge Helix, a proto-spacer adjacent motif (PAM) interacting domain, and two DNA nuclease domains (HNH and RuvC). The REC I domain is responsible for binding to the single gRNA, while the bridge helix is crucial for initiating cleavage activity upon the binding of target DNA [211]. The PAM domain helps in recognizing the consensus NGG (N, any nucleotide; G, guanine) at the 3′ end of the target DNA strand for initiating the binding of the Cas9 to the target DNA [208,212,213,214]. After binding, the Cas9 unzips the DNA to allow the crRNA (within gRNA) to anneal to the target DNA. After annealing, both the strands are cleaved three bases upstream to the PAM, thereby creating blunt ends at the target site. The DSB can be repaired by the error prone NHEJ or HDJ to generate the mutations.

4.2.1. Cas9 Editing

This technique has been beneficial for targeting DNA and RNA viruses with precision and high efficiency (Table 2) [215,216]. Studies using Arabidopsis thaliana or Nicotiana benthamiana showed that single gRNAs targeting the stem-loop sequence within the geminiviral genome or the non-coding intergenic region (IR) showed a stronger interference as compared with those targeting the viral genes like CP or Rep. When gRNAs were used to target non-coding IRs in the Cotton leaf curl Kokhran virus (CLCuKoV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), their systemic movement in the host was blocked [217]. The gRNAs also rendered a broad-spectrum resistance to different mono- and bipartite geminiviruses [218]. The combination of two single gRNAs against the IR and Rep of Cotton leaf curl Multan virus (CLCuMuV) in a single plasmid enhanced the overall resistance of the tobacco plants [219]. CRISPR/Cas9 based approaches have also been used to target the host susceptibility factors such as the eukaryotic translation initiation protein (eIF4E) to improve resistance against many potyviruses [220,221].

Several extensions or modifications of the CRISPR/Cas system have been developed either by mutating the Cas 9 enzyme or by using other variants of Cas. One of the class two effectors, Cas12a (also called Cpf1), has only a RuvC domain and no HNH domain, hence, it generates staggered DSBs instead of blunt [222,223]. The staggered DSB is close to the 3′ end of the target sequence, which creates a 5′ overhang. This could be beneficial for knock-in experiments and also helpful in the insertion of DNA fragments precisely and with perfect orientation through complementary ends by HDR [223]. Also, it requires shorter crRNA than Cas9 and there is no requirement for tracrRNA as per the current reports [224].

4.2.2. Epigenetic Editors

An engineered Cas variant known as dead Cas9 (dCas9) contains inactivated HNH and RuvC nuclease domains [191]. It targets specific genomic regions by recruiting chromatin modifiers and effector proteins, which leads to programmable epigenetic editing [225,226]. dCas9 can be used as a transcriptional activator (CRISPRa) or transcriptional repressor (CRISPRi) [227]. There is enormous potential for dCas9 to gain insights into plant–virus interactions and for performing transcriptional engineering and chromatin alterations [228].

4.2.3. RNA Editors

The FnCas9 (class 2, Type II) derived from Francisella novicida, has RNA-targeting and editing properties [229,230]. The FnCas9 with RNA-targeting gRNA has been used to cleave the ssRNA viruses for resistance against CMV and TMV in N. benthamiana and A. thaliana [231].

Cas13 is a new type of protein isolated from Ruminococcus flavefaciens that comes in class 2 type VI effectors [232]. It has programmable RNA-targeting properties, which broaden the application of the CRISPR/Cas system [215]. Recent studies have demonstrated the effectiveness of using various Cas13 systems successfully for targeting multiple RNA viruses [233]. Transgenic rice and tobacco harboring LshCas13a (Leptotrichia shahii Cas13) with a specific protospacer showed resistance to the Rice stripe mosaic virus (RSMV), TMV, and the Southern rice black-streaked dwarf virus (SRBSDV) [234]. The effectiveness of using LshCas13a to target the helper component proteinase (HC-Pro) and coat protein (CP) segments of the Turnip Mosaic Virus (TuMV) RNA genome in N. benthamiana and A. thaliana, respectively, has been demonstrated. Cas13 has many variants, of which Cas13d is more advantageous than Cas13a, Cas13b, or others [235]. CRISPR techniques, especially CRISPR-Cas12a and CRISPR-Cas13a/d, are also useful in the detection of DNA and RNA virus amplicons generated through isothermal amplification [236].

4.2.4. Base Editors

These are formed by the fusion of the enzyme deaminase with the Cas nuclease domain. Base editors are used to create point mutations without the requirement of a donor template and DSBs [237,238]. This approach is useful for targeting genes responsible for host susceptibility [239]. Cytosine base editors (CBE) and Adenosine base editors (ABE) are two commonly used editors in base editing. The former converts C:G to T:A and the latter converts A:T to G:C [240]. In this process, the availability of the PAM is important to the editing of the target DNA. Advancements in the base editing technology such as variants of CBEs and ABEs are being used to create randomized mutations in targets [241,242]. In Arabidopsis, resistance to Clover yellow vein virus (ClYVV) was obtained by using a novel CBE to convert a susceptible allele of eIF4E1 into a resistance allele through a point mutation of N176K [239].

4.2.5. Prime Editors

Prime editing was achieved by using an engineered nCas9 fused with a reverse transcriptase and prime-editing guide RNA (pegRNA). The pegRNA has a protospacer sequence that directs a Cas nuclease to cleave a target region and add extra sequences, which are responsible for specific changes [243,244]. Like base editing, this technique does not require double-stranded breaks (DSBs). This technique was used to edit PBS1, a decoy host protein, which is responsible for disease resistance. The cleavage of PBS1 by pathogen proteases such as AvrRpt2 from Pseudomonas syringae and Nla from Turnip mosaic virus (TuMV) generates host immune responses. This protein was edited in such a way that it became amenable to cleavage by other pathogen proteases [245]. Hence, precision editing could be used as a promising tool to edit several immune receptors for improving pathogen resistance.

4.2.6. Multiplexing CRISPR/Cas Editing

Targeting multiple sites on a virus in a single go is being developed as an effective strategy to minimize the chances of viral escape. In cotton, three gRNAs were designed to target three viral regions for modifying six overlapping genes (AV2/AV1, AC2/AC3, and AC1/AC4). The analysis showed relatively low virus titers in the plants transformed with all three gRNAs as compared to plants expressing a single gRNA and the wild-type plants [246].

This includes the simultaneous delivery of Cas9 mRNA with multiple gRNAs expression cassettes in one plasmid, artificial gRNAs with Cas9 flanked by Csy4/HH-HDV ribozymes for the cleavage and release of gRNAs, multiple gRNA precursors with Cas9 flanked by tRNAs, etc. [247]. The coupling of CRISPR/Cas13d with an endogenous tRNA system for the expression of four gRNAs was found to be effective against PVY, PVX, PVS, or PLRV viruses in potatoes [248]. The combination of dCas9 with cytosine/adenosine deaminase and an gRNA complex was used for generating point mutations in targets [237,249].

The multiple gRNA strategy is also helpful in the case of mixed viral infections [250]. In potatoes, CRISPR/Cas13a was coupled with an endogenous tRNA system and four different gRNAs to produce a polycistronic tRNA-gRNA (PTG) against PVY. However, the transgenic lines exhibited similar levels of tolerance as observed in the case of the single gRNA lines, indicating that multiple target sites did not affect viral interference [248].

The major hurdles to the GE approaches are the generation of off-targets due to the constitutive expression of the Cas nuclease or sequence mismatches in gRNA [179]. The error-prone repairing process creates selection pressure on viruses which results in a generation of viral variants having a resistance towards editing. The replacement of a constitutive promoter by a virus inducible promoter for the expression of the Cas nuclease is quite useful for bringing down the off-targets [251].

Table 2. List of GE approaches available against Plant DNA and RNA viruses.

Strategy	Virus	Target	Plant	References
For DNA Viruses
ZFN	Tomato yellow leaf curl China virus, Tobacco curly shoot virus	AC1 gene	Tobacco	[193]
	Bean yellow dwarf virus	Cis-acting long and short intergenic region		[252]
TALENs	Tomato yellow leaf curl China virus, Tobacco curly shoot virus, Tomato leaf curl yellow virus	Motif 1 and 2 of AC1 ORF	Tobacco	[204]
AZP	Beet severe curly top virus	Rep protein	Arabidopsis	[194,253,254,255]
	Tomato yellow leaf curl virus
	Rice tungro bacilliform virus	RTBV promoter		[256]
CRISPR/Cas9	Cotton leaf curl Kokhran virus	CP and Rep	Tobacco	[217]
	Merremia mosaic virus Tomato yellow leaf curl virus	Intergenic region
	Bean severe curly top virus	Viral genome	Tobacco	[257]
	Bean yellow dwarf virus	Rep, short and long intergenic region	Tobacco	[258]
	Chilli leaf curl virus	Intergenic region and overlapping (V2/V1) and C1/C4)	Tobacco	[259]
	Cauliflower mosaic virus	CP gene	Arabidopsis	[260]
	Wheat dwarf virus	MP, CP, Rep/RepA, long intergenic	Barley	[261]
	Banana streak virus strain Obino I’Evai African cassava mosaic virus	ORF1, ORF2 and ORF3 AC2 and AC3	Banana Cassava	[262] [263]
For RNA Viruses
MNs	Tobacco rattle virus	Dihydroflavonol 4-reductase (DFR)	Tobacco	[264]
FnCas9	Tobacco mosaic virus, Cucumber mosaic virus	ORF1a/2a/2b/3a/CP	Tobacco	[231]
	Cucumber mosaic virus		Arabidopsis
LshCas13a	Turnip mosaic virus	HC-Pro	Arabidopsis	[216]
	Southern rice black-streaked dwarf virus	Viral dsRNA	Rice	[234]
	Rice stripe mosaic virus	Viral ssRNA
Cas13d/PTG	Potato virus X, Potato leafroll virus, Potato virus Y, Potato virus S	CP	Potato	[248]
Cas13a/PTG	Potato virus Y	P3, CI, NIb and CP	Potato	[265]
Cas13a with multiplex gRNAs	Potato virus Y	PI, HC-Pro, P3, Cl, Cl2, VPg	Potato	[266]
LbCas12a	Cotton leaf curl Multan virus	C1, C2/C3, V1	Cotton	[267]
Cas12f	Cotton leaf curl Kokhran virus and Cotton leaf curl Multan virus	CP	Cotton	[268]

Table 2. List of GE approaches available against Plant DNA and RNA viruses.

Strategy	Virus	Target	Plant	References
For DNA Viruses
ZFN	Tomato yellow leaf curl China virus, Tobacco curly shoot virus	AC1 gene	Tobacco	[193]
	Bean yellow dwarf virus	Cis-acting long and short intergenic region		[252]
TALENs	Tomato yellow leaf curl China virus, Tobacco curly shoot virus, Tomato leaf curl yellow virus	Motif 1 and 2 of AC1 ORF	Tobacco	[204]
AZP	Beet severe curly top virus	Rep protein	Arabidopsis	[194,253,254,255]
	Tomato yellow leaf curl virus
	Rice tungro bacilliform virus	RTBV promoter		[256]
CRISPR/Cas9	Cotton leaf curl Kokhran virus	CP and Rep	Tobacco	[217]
	Merremia mosaic virus Tomato yellow leaf curl virus	Intergenic region
	Bean severe curly top virus	Viral genome	Tobacco	[257]
	Bean yellow dwarf virus	Rep, short and long intergenic region	Tobacco	[258]
	Chilli leaf curl virus	Intergenic region and overlapping (V2/V1) and C1/C4)	Tobacco	[259]
	Cauliflower mosaic virus	CP gene	Arabidopsis	[260]
	Wheat dwarf virus	MP, CP, Rep/RepA, long intergenic	Barley	[261]
	Banana streak virus strain Obino I’Evai African cassava mosaic virus	ORF1, ORF2 and ORF3 AC2 and AC3	Banana Cassava	[262] [263]
For RNA Viruses
MNs	Tobacco rattle virus	Dihydroflavonol 4-reductase (DFR)	Tobacco	[264]
FnCas9	Tobacco mosaic virus, Cucumber mosaic virus	ORF1a/2a/2b/3a/CP	Tobacco	[231]
	Cucumber mosaic virus		Arabidopsis
LshCas13a	Turnip mosaic virus	HC-Pro	Arabidopsis	[216]
	Southern rice black-streaked dwarf virus	Viral dsRNA	Rice	[234]
	Rice stripe mosaic virus	Viral ssRNA
Cas13d/PTG	Potato virus X, Potato leafroll virus, Potato virus Y, Potato virus S	CP	Potato	[248]
Cas13a/PTG	Potato virus Y	P3, CI, NIb and CP	Potato	[265]
Cas13a with multiplex gRNAs	Potato virus Y	PI, HC-Pro, P3, Cl, Cl2, VPg	Potato	[266]
LbCas12a	Cotton leaf curl Multan virus	C1, C2/C3, V1	Cotton	[267]
Cas12f	Cotton leaf curl Kokhran virus and Cotton leaf curl Multan virus	CP	Cotton	[268]

5. Summary and Future Perspectives

Viruses invade plant cells and hijack the host’s cellular machinery for their own multiplication. The plants fight back by activating cellular defense responses resulting in genetic and epigenetic events to block viral gene expression. Several attempts were made to introgress resistance to a virus in cultivated plants by breeding with its wild relatives, but were unsuccessful. Subsequently, strategies were changed to directly target the virus for conferring resistance. The GM and GE approaches for targeting the virus offered great promise in saving crops from viral infection (Figure 1).

The early GM approaches were based on engineering PDR by over-expressing CP or other viral proteins, and it proved to be effective against many viruses. However, with the discovery and understanding of the underlying principles of RNA silencing and its role in providing resistance to plant viruses, there was a paradigm shift in these strategies. The first silencing tools employed the dsRNA or hpRNA precursors directed against specific viral genes. The transgenic papayas expressing siRNAs against the Papaya ringspot virus (PRSV) proved to be a boon for the Hawaiian papaya industry. The EMBRAPA 5.1 bean was the first RNA silencing-based GM crop approved and cultivated in Brazil [269]. It was developed by transforming plants with the AC1 gene of the Bean golden mosaic virus. The RNAi-based cassava showing resistance to the Cassava brown streak virus was also approved for release by the Kenya national Biosafety authority (NBA). GM technology has helped to save the papaya grown in Hawaii and reduce potato crop losses in the USA and Canada. These case studies pointed out the potential of genetic engineering for generating strong resistances against plant viruses.

The siRNAs had the limitations of being less specific and generating undesirable off targets [270]. This resulted in the development of amiRNA tools by engineering the natural pre-miRNA backbones. These second sets of tools were more specific but had a limited targeting capacity, so syn-tasiRNA vectors were favored and developed as the third set of tools. However, the skepticism regarding the acceptance of GM crops by the public has hindered the active implementation of this technology. Regulations of GM crops vary from country to country, e.g., the USA focusses on the risk assessment of the ultimate food product rather than the process by which it was made [271]. With the help of deep sequencing, a wide range of endogenization of viral genomes was observed in plants which clearly suggests the incorporation of foreign genes is not that unnatural [272]. Also, there is still a lack of evidence in support of claims of the adverse effects of GM crops on humans and animals [273].

As an alternative strategy, the search for an exogenous application of dsRNA precursors or small RNAs to activate the host-silencing pathways was started (

Table 3. List of exogenously delivered dsRNAs for the induction of RNA silencing against plant viruses.

dsRNA Coating and Inoculation	Virus	Target	Plant	References
LDH nanosheets (amiRNA)	Cucumber mosaic virus	AV1 and AV2	Tomato	[275]
Clay nanosheets (dsRNA)	Tomato yellow leaf curl virus	2b suppressor Replicase	Tobacco	[270]
Agroinfiltration and Direct spray	Cucumber green mottle mosaic virus Tomato leaf curl new delhi virus	AC1 and BC1	Cucumber	[276]
High pressure spraying of free siRNAs	Cauliflower mosaic virus	CaMV 35S promoter	Tobacco	[277]
Mechanical inoculation of free dsRNAs In vivo and in vitro synthesized dsRNAs	Tomato mosaic virus Cymbidium mosaic virus Cucumber mosaic virus	CP and MP CP CP and 2b	Tomato Orchid Tobacco Chenopodium Quinoa	[274,278] [279]
CQAS-dsRNA nanoparticles and Carbon Quantum Dots (CQDs) through Root soaking, infiltration	Potato virus Y	CP	Tobacco Tomato Pepper	[280]

). Several reports have highlighted the use of high pressure (4–6 bar) for the efficient transfer of dsRNA in the nucleus for spray-induced epigenetic modifications (SPIEM). When high pressure spraying was used to transfer 333 bp of dsRNA in N. benthamiana, there was no induction of methylation at the target region at three days post spraying (dps), while 28% of samples were methylated at six dps and 40% of samples were methylated at ten dps [274]. However, spraying with low pressure did not result in any methylation. The RNA-based bioformulations are in various stages of development and are anticipated to prove useful in tackling viral infections.

The emergence of GE tools has added another arsenal in the fight against viruses infecting crop plants. CRISPR/Cas9 provides a robust system for generating more effective and prolonged resistances against viruses. The recent additions of Cas13 and Cas12 variants have proved very effective in targeting RNA viruses. The efficacy of CRISPR-based approaches primarily depends on the method of delivery and the design of the gRNA sequence. A number of delivery methods including Agrobacterium-mediated T-DNA transformation, protoplast transfection, microprojectile bombardment, and virus-based methods have been deployed to introduce CRISPR/Cas components into plants. As viruses have the tendency to mutate at a faster rate, multiplexing technologies can contribute to precise editing and broadening the range of the targeted viruses. Although the use of CRISPR/Cas for the development of viral-resistant plants has been demonstrated to be effective, further improvements in the technology are awaited to attain efficient, durable, and broad-spectrum resistance.