Application of Reverse Genetics in Functional Genomics of Potyvirus

Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus–host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.


Introduction
Reverse genetics of RNA viruses refers to the generation of recombinant viruses through site-directed mutagenesis such as substitution, deletion or insertion [1]. This consequently made various phenotypic studies possible, apart from providing a powerful tool to enhance our knowledge on life cycles and pathogenic mechanisms of RNA viruses as well as the structure or function of individual viral genes [2,3]. Besides that, reverse genetics approaches have largely contributed to the development of antiviral therapeutics, vaccines [4,5], and vectors [6]. The first success in reverse genetics for an RNA virus was reported for poliovirus, a positive-stranded RNA virus in 1981 [7]. Since then, reverse genetics technology has greatly revolutionized the studies on almost every group of positive-strand viruses including potyviruses [8].
Potyvirus is a genus of virus that belongs to the family Potyviridae. Potyviruses are among the most economically important and widely spread groups of plant viruses [9]. The genus comprises 158 species including Potato virus Y as the type species [10]. The picornavirus-like supergroup [11] is transmitted non-persistently by aphids. However, some potyviruses are also transmitted through seeds [12]. Potyviruses have a single-stranded RNA genome of around 10 kb, with a 3 terminal poly(A) tail and a VPg protein at its 5 end [13]. The members of potyvirus group exist as flexuous rods ranging from 720 to 900 nm in length [14]. The single open reading frame (ORF) in the RNA molecule is translated into a large polyprotein. Proteolytic cleavage of the polyprotein by three viral proteinases [15,16] gives rise to ten functional proteins as follows: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP [17,18]. Additionally, the expression of a P3N-PIPO protein has been recently exhibited as a result of either a transcriptional slippage or ribosomal frameshift [19,20].
Infectious clone technology represents the most commonly applied reverse genetics system. Cloning of an infectious clone was first described for Brome mosaic virus [21]. Since then, infectious clones for many other plant RNA viruses were successfully obtained as either in vivo or in vitro trancripts. For in vitro strategy, the viral cDNAs are cloned under bacteriophage promoters such as T7, T3, or Sp6 followed by the generation of in vitro transcripts [22,23]. On the other hand, in vivo infectious transcripts are driven by Cauliflower mosaic virus 35S promoter in a binary vector. Cells transformed with the plasmids containing virus cDNA clones are then introduced into plants through agroinfiltration, particle bombardment, or rubbing onto the leaf's surface [24,25]. In this review, we describe and summarise the application of reverse genetics in potyviral studies based on the potyvirus infectious clones developed to date.

Point Mutation
Point mutation refers to a change occurred within a gene due to a single base pair alteration in the DNA sequence. During translation, conversion of RNA copied from the DNA into a sequence of amino acids would take place and point mutations usually cause a variety of effects on the protein synthesized at the final stage [26].

Effect of Point Mutation on Virus Biological Properties
Mutagenesis studies aimed to characterize the phenotypic consequences resulting from genome modification, based on how the progenies differ from their respective wild type [27]. To date, various site-specific mutations in the potyviral genomes and screening of the progeny viruses have been conducted (Table 1). For instance, a 10-fold reduction in virus accumulation could be observed when a single point substitution, serine to glycine at position 7 was introduced in the DAS motif of CP N terminus of B11 isolate of Potato virus A (PVA). However, aphid transmissibility of the virus was still retained [28]. Further information on potyviral noncoding region (NCR) roles were gathered by studying the changes observed in biological characteristics of mutants with manipulated NCR. In line with that, Clover yellow vein virus, pClYVV cDNA clones containing no poly(A), poly(A) with shorter A residues, or various oligonucleotide sequences downstream were constructed. All RNA progenies obtained from the pClYVV cDNA constructs had poly(A) tails with infectivity varies along the sequences introduced. This suggested the addition of poly(A) to be independent of template, yet essential to maintain the infectivity of potyviral cDNA [29]. Besides that, a deletion mutation introduced in the NCR at 5 end of Plum pox virus (PPV) revealed that the region between nucleotides 39 and 145 contributes to competitive fitness of the viral population [30]. A point mutation engineered into Turnip mosaic virus, TuMV-pXBS7/TuMV-pXBS8 chimeric viruses have allowed the mapping of a symptom severity determinant in the 3 -terminal UTR of pXBS8-derived virus [31]. [32] Tobacco etch virus TEV pTEV7D-GUS contains site-directed mutagenesis in GUS-HC-Pro fusion protein: • TEV-2del r retained nucleotides GUS 1 →GUS 135 but lost HC-Pro sequence up to nucleotide 207 • TEV-7del r retained nucleotides GUS 1 →GUS 9 but lost HC-Pro sequence up to nucleotide 265 The sequence deleted from TEV-2del r and TEV-7del r composed the N-terminal domain and a cysteine-rich motif of HC-Pro. The ability of these mutants to replicate and move systemically indicated that the N-terminal domain of HC-Pro is not a factor essential for these processes. Although both TEV-2del r and TEV-7del r were viable in plants, a negative effect on accumulation of viral RNA and coat protein could be observed, suggesting a potential function of HC-Pro in enhancing viral replication. [11] Plum pox virus PPV-D PPV mutant that lacked long sequences located between nucleotides 39 and 145 in 5 NCR (146 nt long) The deleted region is not necessary for genomic RNA replication, but contribute to the competitive fitness of the PPV since the mutants were not able to compete with the wild-type strain in co-inoculation experiments. [30] PPV mutant without sequences between nucleotides 127 and 145 in 5 NCR Plant infected with PPV mutant viruses ∆[127,145] showed a very mild symptoms. However, the wild-type symptom severity was recovered after spontaneous second-site mutations.
pClYVV 3 dup 1: Last 5 nts, CGAGA was duplicated pClYVV 3 dup 4: Last 10 nts, TAGAGCGAGA was duplicated The infectivity of pClYVV 3 dup 4 was higher than that of pClYVV 3 dup 1. This suggested that the length of duplicated sequence (downstream of the poly(A) site) might have enhanced mutants' infectivity. Long-term passage of progenies of molecularly cloned SMV strain G7 in Rsv1-genotype soybean resulted in an emergence of a mutant, SMV-G7d. A total of seven amino acid substitutions in SMV-G7d genome lead to its incapability in provoking either Rsv1-mediated lethal systemic hypersensitive response or PR-1 protein gene transcript upregulation as parental SMV-G7 and thus evade an R-mediated recognition. [37] Soybean mosaic virus Amino acid substitution of N 286 →D 286 in HC-Pro of SMV A297-12 The change N286D reduced silencing suppressor activity of SMV A297-12. [38] SMV A297-12,SMV A297-13, SMV 413 Substitution of the HC-Pro in SMV 413 infectious clone with that of:

Role of Point Mutation in Cross Protection Phenomenon
Cross protection is conferred by pre-infecting the host crops with a mild virus strain to prevent the infection by another severe or closely-related strain of that virus [40]. This strategy is a promising biological method to control plant viruses [41]. However, the availability of natural attenuated strains of plant viruses is limited [42]. Hence, point mutations were intentionally engineered into plant viruses for the development of less virulent strains that could be utilized in cross protection [43]. For example, a lysine in the IDEKK motif and glycine in the HC-Pro C terminus of Papaya ringspot virus (PRSV) were changed to aspartic acid and lysine respectively. Consequently, PRSV mild mutants with a potential to cross-protect Cucumis melo plants against wild type PRSV-W were obtained [44]. Similarly, attenuated isolate M11 of Bean yellow mosaic virus (BYMV) was generated by exchanging an amino acid within the large ORF, leucine with serine. BYMV-M11 mutant conferred a complete cross protection against BYMV isolates from gladiolus, incomplete against BYMV isolates from other hosts, partial against a CIYVV isolate [41]. An arginine and a glutamic acid at positions, 180 and 396 in the Zucchini yellow mosaic virus (ZYMV) HC-Pro were substituted with isoleucine and asparagine respectively. The attenuated mutants were found to induce only mild symptoms with recovering and protected squash plants completely against the severe Taiwan strain, ZYMV TW-TN3 [42]. Another mild isolate of ZYMV, ZYMV-AG, was also generated through a substitution of isoleucine for arginine within the conserved FRNK motif of HC-Pro. Cucurbit plants inoculated with ZYMV-AG mutant were protected against the infection by severe ZYMV-NAT and ZYMV-CA isolates [45].

Virus/Host Interaction
Mutagenesis studies on potyvirus infectious clones allowed the mapping of viral determinants responsible for viral genome replication, local and systemic movement of virus, symptomatology and host species range [46] (Table 2). This knowledge facilitates the understanding of complex processes underlying interactions between viral and host factors during a viral infection in plants [1].

Viral Determinants
The differential infectivity of UK 1 and JPN 1 strains of TuMV in Ethiopian mustard was studied to map the viral determinants involved [47]. Isolate UK 1 causes a systemic infection in the host while JPN 1 does not. UK 1 and JPN 1 recombinant viruses were made by exchanging the amino acids found in one isolate to that in the other isolate at several positions within the P3 C-terminal domain, leading to the identification of two adjacent positions (1099 and 1100) in TuMV-JPN 1 as the main resistance determinants. GFP-tagged viruses were also constructed to analyse the resistance of Ethiopian mustard to isolate JPN 1. Consequently, both inoculated and non-inoculated leaves showed a virus-induced fluorescence in separate areas, indicating that the non-host resistance is only apparent. In another previous study, the NIb protein of Potato virus Y, PVY-SON41 was demonstrated as the avirulence factor corresponding to Pvr resistance gene in pepper. A single substitution of adenosine nucleotide to guanosine at position 8424 in that region is sufficient for the virulence [48]. Table 2. List of molecular determinants mapped in potyviral genomes through reverse genetics method.

Virus Genome Manipulation Application Findings Reference
Turnip mosaic virus TuMV Chimera pXBS78 derived from pXBS7 contains a fragment of 8975-9311 residues from 3 terminal UTR of pXBS8 Chimera pXBS87 derived from pXBS8 contains a fragment of 8975-9253 residues from 3 terminal UTR of pXBS7 Genetic determinant (symptom severity) pXBS7 induced symptoms in infected tobacco plants that are indistinguishable from those produced by native TVMV RNA. In contrast, pXBS78 induced only very mild barely detectable symptoms in infected plants. The results of sequence analysis and genome exchange experiments indicate that a 58-nt segment consisting of patterns of adenine and uracil residues in the 3 UTR of pXBS8-derived RNA is responsible for the symptom attenuation phenotype. [31] Exchange of 5 UTR, P1pro and HCpro of P-1 with the corresponding regions from P-4 creating: vP-1(P-4 5 UTR) vP-1(P-4 P1pro) vP-1(P-4 HCpro) respectively Genetic determinant (seed transmission) P-1 is highly seed-transmitted whereas P-4 is rarely seed-transmitted. The seed transmission frequencies of vP-1(P-4 5 UTR) and vP-1(P-4 HCpro) were reduced to 50% and 20% of vP-1, respectively, while vP-1(P-4 Plpro) was seed transmitted at the same frequency as vP-1. This showed that the HC-Pro was a major determinant of seed transmission while the P1pro showed no measurable influence. The region encodes for C-terminal part of P3+6K 1 , differ at 11 positions between PPV-R and PPV-PS and contains all information required to transform the R-type into PS-type symptomatology in Nicotiana clevelandii.

[13]
Potato   LMV-E could overcome the protection afforded by the resistance genes mo1 1 or mo1 2 , resulting in systemic mosaic symptoms whereas LMV-0 could not. HC-Pro of LMV-E is responsible for causing severe stunting and necrotic mosaic in susceptible cultivars. In contrast, the ability to overcome mo1 resistance was mapped to the 3 half of the LMV-E genome. [56] Turnip mosaic virus TuMV-UK1

Host Specificity
The differential responses of UK 1 and JPN 1 strains of TuMV in different hosts; brassicas (Ethiopian mustard, Indian mustard, turnip) and radish cultivars (Icicle, round red radish with a white tip (RRRWWT), Daikon) were investigated [62]. Although both infectious clones caused infection in all three brassicas, the symptoms induced were observed to be different from each other. In the case of radish, TuMV-JPN 1 infected European types (Icicle & RRRWWT) but not Japanese Daikon radish. In contrast, TuMV-UK 1 was not able to infect any radish type, showing its distinct biological properties compared to UK 1 isolate. In addition, two more isolates of TuMV, Tu-2R1 (pTuR1) and Tu-3 (pTuC) also exhibited distinct host-specific infection phenotype and symptomatology in cabbage and radish [63]. Tu-2R1 systemically infect Japanese radish, inducing mosaic symptoms apart from very mild chlorotic mottle in cabbage (Brassica oleracea L.). Meanwhile, Tu-3 induced systemic chlorotic and ringspot symptoms in infected cabbage but not radish. The genomic region encoding C terminus of HC-Pro, all of P3 and N terminus of 6K1 was exchanged between pTuC and pTuR1 chimeras. The only difference between pTuR1 and pTuC was identified within the amino acid sequence of P3 gene. Hence, P3 gene was suggested to be involved in the differential phenotypes caused by TuMV isolates in both hosts.
Host specificity of two different Johnsongrass mosaic virus (JGMV) strains, Johnsongrass infecting JGMV-Jg and Krish strain JGMV-Kr, was studied. Transcripts of JGMV-Jg full-length chimera containing coat protein sequences from JGMV-Kr were infectious in Krish resistant sorghums, thus confirming the role of JGMV-Kr coat protein in its host specificity [64]. Similarly, PRSV strain belongs to type p (PRSV P) possessed different biological characteristics compared to type W (PRSV W). Host species range of PRSV W is limited to Chenopodiaceae and Cucuribitaceae families whereas PRSV P infects plants in papaya family (Caricaceae) additionally [65]. Host assays using recombinant viruses generated between PRSV P-YK and PRSV W-CI in combination with site-directed mutagenesis revealed that lysine amino acid at position 27 in NIaPro acts as the host specificity determinant in PRSV for infecting papaya [66]. A single amino acid mutation, either lysine to aspartic acid or vice versa at position 27 is sufficient for the switching of PRSV host range between non-papaya infecting and papaya-infecting respectively [67].
The experimental plants were agroinoculated with an infectious clone of RU1 strain, pCA-RU1-GUS containing UidA gene. In situ histochemical GUS assays revealed that DW, The Prince, CRM, SP, and SGR cultivars (carrying bc-u resistance gene) were all systemically infected. However, the genotypes carrying the I gene alone (Widusa) or in combination with bc-1 (Topcrop, ITG), bc-12/bc-22 (IVT-7233), and bc-3 (USCR-7, Raven) showed only a weak blue staining. These resistance responses against BCMV-RU1 are largely in agreement with previous results obtained through immunological method and symptom descriptions based analysis [69]. The study suggested that resistance gene I conferred a complete protection against BCMV-RU1 while bc-genes do not provide any. Besides that, the host defence response of cucurbit genotype, Dina-1 against ZYMV-NAA potyvirus has also been focused on previously [70]. Switches in amino terminus of the virus coat protein breaks Dina-1 resistance against ZYMV-NAA, suggesting its resistance gene product to be involved in direct interaction with the substituted region.
To learn more on mechanisms of interrelation between potyvirus and host, the impact induced by TuMV infection on the endomembranes of host early secretory pathway was examined [71]. TuMV infection caused the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts being amalgamated as a perinuclear globular structure that included viral protein, 6K 2 vesicles too. TuMV 6K 2 fused to photoactivable GFP (PAGFP) was used to monitor the vesicle movement. Viral egress is shown to begin with the budding of 6K 2 vesicles at ER export site in the globular structure. The virus then travels to plasma membrane and plasmodesmata to be delivered into neighboring cells. Some peripheral vesicles were also observed to be recycled back to the globular structure. This indicated a functional linkage between the peripheral vesicles and perinuclear structure. Although the Golgi apparatus and ER lost their organization due to the amalgamation, they were still connected to the host secretory pathway (ER-to-Golgi transport). The importance of this connection was investigated by inhibiting the pathway. As a result, the disruption enhanced the clustering of peripheral 6K 2 vesicles with COPII coatamers, leading to an inhibition of cell-to-cell movement of virus. This suggests the requirement of a functional secretory pathway for a successful intercellular propagation of TuMV.
Along with these host-viral factor interactions, the interplay between Tobacco etch virus (TEV) and Arabidopsis thaliana proteins was also investigated [72]. An affinity polypeptide Twin-Strep-tag (TST) was inserted between codons of VPg and NIaPro domains in the TEV NIa to facilitate the study. A total of 232 different Arabidopsis thaliana proteins targeted by viral proteins were identified through affinity purification followed by mass spectrometry analysis (AP-MS). VPg and NIaPro specifically targeted 89 and 76 of these proteins, respectively. Overall, a total of 67 proteins targeted by both domains were considered to be the targets of full-length NIa.

Viral Proteins
Apart from the mature proteins, proteolytic processing of the potyviral polyprotein also produced multiple partially processed intermediates [73]. The different potyviral proteins act in a coordinated and interdependent manner. About 33 interactions were identified between potyviral proteins through a testing of 58 protein combinations in planta [74,75]. This broad network of interrelations with different viral and host proteins contributes to the multifunctional nature of potyviral proteins [76].

Functional Importance
Potyviral HC-Pro and CP proteins were reported to have a crucial role in efficient aphid transmission [28]. In line with that, a change of lysine amino acid to glutamic acid at the position 307, within Zinc-finger motif of HC-Pro completely abolished insect transmission activity of Tobacco vein mottling virus, TVMV-WT. The mutation suggested might have altered the motif structure and thus the binding property of HC-Pro too [32]. Likewise, a TEV mutant, TEV-2del r lacking 207 nucleotides in HC-Pro sequence exhibited an aphid-non-transmissible phenotype. However, its transmission activity was restored partially by pre-feeding the aphids on active HC-Pro from PVY. This confirms the helper activity of the N-terminal domain of HC-Pro [11]. A mutation introduced within CDNQLD motif of ZYMV-A HC-Pro also resulted in an almost complete absence of symptoms and partial reduction of viral accumulation [77]. The motif sequence was suggested to be involved in symptomatology or silencing inhibition. On the other hand, when glutamic acid at amino acid position 68 within the CP of PVY-N605 was substituted with a lysine, aphid transmission of the virus increased by two folds [35].
Seo et al. [78] conducted a yeast two-hybrid system (YTHS) and galactosidase assays to investigate the interaction between CP and HC-Pro in Soybean mosaic virus, SMV-G7H. A highly conserved histidine in the CP C-terminus and an arginine near the cleavage site at HC-Pro C-terminus were mutated and the results obtained showed that both amino acids are necessary to maintain the interaction for a successful transmission of SMV by aphids. Moreover, an amino acid substitution in the DAG motif was found to have disrupted the CP-HC-Pro interaction in YTHS.

Structural Importance
In order to figure out the function of 3 -UTRs of potyviruses, the sequences between nucleotide positions 8-42 in the 3 UTR of a Tobacco vein banding mosaic virus, TVBMV-HN39 infectious clone, pCaTVBMV-GFP were deleted. As a consequence, the mutant caused no systemic infection in inoculated Nicotiana benthamiana plants. According to the RNA secondary structures prediction, the deleted region is able to form a stem-loop (SL) like structure. Progenies derived from TVBMV mutants lacking nucleotides between positions 1 and 20 and 15 and 35 within 3 -UTR were found to have restored the 5 -end SL like structures and systemically infected tobacco plants. Hence, the 5 -terminal stem loop was proposed to be neccessary for TVBMV systemic infection [79]. Formation of the stem-loop structure by a conserved nucleotide motif in 3 UTRs of 15 potyviruses including the New Zealand isolate of Clover yellow vein virus (CYVV-NZ) has been reported previously [80].

Viral Vectors
Apart from bacterial and yeast expression systems, the plant viral vectors also provide a fast and efficient approach for synthesis of specific proteins in plant cells [81]. In this context, potyviruses have often been used as gene expression vectors due to some of their advantageous traits [12]. For instance, their rod shape make them less restrictive to accommodate large genome inserts [82]. Furthermore, it is well-known that potyviruses infect all types of plant tissues including seeds [83][84][85]. Two different insertion sites in potyviruses were exploited for the introduction of target genes, either between P1 and HC-Pro or else between NIb and capsid protein cistrons [86]. These criteria allowed a simultaneous expression of two foreign proteins [87], either as free molecules or fused to viral proteins [88,89].

Gene Tagging
Viral vectors are usually tested through an expression of well-analyzable reporter genes in plants [46]. With reference to that, a ZYMV full-length clone containing GUS gene under a Strawberry vein banding virus (SVBV) viral promoter was inoculated into experimental host plants [90] (Table 4). The GUS gene was found to be expressed stably in infected tobacco plants, indicating the applicability of ZYMV infectious clone as a viral vector and the functionality of the novel SVBV promoter in driving ZYMV infection. Apart from that, tagging a TEV clone with GUS marker gene eased the monitoring of virus replication and spread following infection through a simple histochemical assay in situ [91] ( Table 4).

Expression of Biologically Active Polypeptide
The potential of Brome mosaic virus (BMV) to be developed into a plant virus vector was successfully demonstrated in 1986 [92]. Since then, massive efforts have been taken in constructing vector systems with plant viruses for the expression of foreign genes in planta ( Table 3). The main goal of plant genetic modifications by incorporating transgenes is to change crop properties, leading to increased yields or higher quality of the agricultural products. For instance, a soybean glutamine synthetase (GS) together with GFP were expressed using a CIYVV-vector system, resulting in glufosinate herbicide tolerance and early flowering of legume plants [84]. In a similar way, the expression of endoglucanase D (EngD) in N. benthamiana using a Pepper mottle virus, PepMov-based vector led to an increased senescence along with milder symptoms [93]. Another purpose of developing transgenic plants is to produce different foreign substances of protein nature [46]. For an example, the nucleocapsid proteins (NPs) of tospoviruses were expressed by a ZYMV vector in squash plants. Those NPs act as immunogens for the production of highly specific polyclonal antiserum and monoclonal antibody [81]. Likewise, a transcription factor, Rosea1 tagged infectious clone of PVY was developed, conferring benefits to molecular farming by rapidly produced larger amounts of anthocyanins in biofactory crops [94]. [88] Zucchini yellow mosaic virus ZYMV TW-TN3 ZWBNV-N recombinant contains nucleocapsid protein (NP) ORF of Watermelon bud necrosis virus inserted between the P1 and HC-Pro.

Expression of antigen
Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. The ZYMV-expressed WBNV NP was purified from extracts of the infected squash plants and was used as an immunogen for production of specific antiserum in a rabbit and monoclonal antibodies in mice. [81] Papaya ringspot virus PRSV-W pVD2EDIII contains a histidine tagged dengue E protein domain III (DENV 2 E) inserted between the P1 and HC-Pro

Expression of antigen
The construct was designed to generate a discrete antigen moiety (D2EDIII) after proteolytic processing. However, the E protein insert was fused to the PRSV P1 protein, suggesting inefficient protease processing at the P1/D2EDIII junction. Despite the failure, the insert was shown to be stable over 2 passages PRSV indicating the vector suitability and stability for the expression of heterologous proteins in zucchini plants. [103] Potato virus A PVA-B11 PVA vectors containing soluble resistance-related calcium-binding protein (sorcin) catechol-O-methyltransferase (S-COMT) between NIb and CP

Expression of genes human origin
The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. S-COMT with high levels of enzymatic activity were produced. However, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein.
[104] Female mice were orally treated with the vDer p 5 extract. As a result, the allergen inhibited Der p 5-specific IgE synthesis and airway inflammation, clinically relevant to human asthma. This provides a novel approach for the therapy of allergic asthma. [106] Soybean mosaic virus SMV-G7H RNA silencing suppressors 2b and p19 genes were cloned into pSMV-MCS between the P1 and HC-Pro cistrons (pSMV-2b and pSMV-p19 respectively)

Expression of RNA silencing suppressors
Severe symptoms including stunting, extensive leaf deformation and shrivelling was detected in either pSMV-2b or pSMV-p19 infected soybean, with the accumulation of SMV RNAs and CP similar to that in plants infected with pSMV-GFP. [87] Partial forward sequence (567 bp) and partial reverse sequence (282 bp) of were inserted into pSMV-MCS between the P1 and HC-Pro cistrons (pSMV-spPDSfw and pSMV-spPDSrv respectively)  In addition to the NT regions of HC-Pro and CP, the NT regions of P3, CIP and NIb were also able to carry both heterologous ORFs to be translated as a part of the polyprotein and processed as free-form protein although showed more permissiveness to the GFP ORF than Der p 5 ORF. The efficiency and stability of expression of the ORFs depends on the particular ORF and the host plant employed.

Tobacco etch virus TEV
A series of six histidines (his-tag) inserted near the 5 terminus of the HC coding region in pTEV-HCHXa pTEV-HCHXa was infectious, produced symptoms in tobacco similarly as wild-type TEV, and stably maintained through at least 4 cycles of aphid transmission. HC protein purification based on the affinity of its his-tag for Ni2+-charged resin, yielded large amount of fully functional his-tagged HC protein. [97] Clover yellow vein virus CIYVV pClYVV-GFP contains a gfp gene inserted between P1 and HC-Pro. Junctions between the inserted proteins contained the protease cleavage recognition sites Green fluorescence was detected in broad bean, kidney bean, and soybean plants infected with pClYVV-GFP. The stability of the construct in the symptomatic tissues was confirmed by RT-PCR and Western blot analyses. [84] Plum pox virus PPV-D pICPPV-NK-GFP contains a gfp gene inserted between the NIb and CP junction GFP was detected in crude extracts from PPV-NK-GFP infected leaves by Western blot. Genetic stability of the chimera was confirmed by IC-PCR amplification of a cDNA fragment including the foreign sequence of expected size. Virus and GFP accumulations were quantified in infected N. clevelandii plants by ELISA. [88] Soybean mosaic virus SMV-G7H GFP cloned into pSMV-MCS between the P1 and HC-Pro cistrons (pSMV-GFP) Typical mild mosaic symptoms and systemic expression of GFP protein were detected in pSMV-GFP infected soybean. The GFP gene was shown to be maintained stably in soybeans even after three serial passages. [87] Tobacco etch virus TEV pTEV7D contains a β glucuronidase (GUS) gene between the polyprotein-coding sequences for N-terminal 35-kDa proteinase and HC-Pro GUS act as a marker gene in TEV genome, demonstrating that virus replication and movement can be monitored easily by using a simple histochemical assay in situ. The GUS enzyme was proteolytically excised as a fusion product with HC-Pro. [91] Lettuce mosaic virus LMV-E pLMVE-GFP and pLMVE-GUS contains a jellyfish GFP & β glucuronidase (GUS) gene respectively, fused to HC-Pro Both GFP-and GUS-tagged viruses induced attenuated symptoms in susceptible lettuce cultivars Trocadero and Vanguard, compared to wild-type. Accumulation of the recombinant viruses was either undetectable (pLMVE-GUS) or strongly delayed and inhibited by 90% (pLMVE-GFP). In contrast to parental virus, the recombinants were unable to overcome the resistance gene, mo12. [83] Pepper mottle virus PepMoV-Vb1 SP6PepMoV-Vb1/GFP contains turboGFP inserted between NIb and CP coding regions Expression of GFP was monitored under illumination. SP6PepMoV-Vb1/GFP was highly infectious and symptoms were not different from those induced by either pSP6PepMoV-Vb1/wild-type PepMoV-Vb.

Virus Genome Manipulation Findings Reference
Papaya leaf distortion mosaic virus PLDMV-DF pPLDMV-GFP and pPLDMV-mCherry contain a GFP and mCherry into the NIb/CP junction respectively PLDMV-GFP or PLDMV-mCherry developed typical systemic symptoms in 95% of infected papaya seedlings, in which fluorescence was observed in leaves, stems, and roots. Both clones were stable in papaya for more than 90 days and during six serial passages at 30-day intervals. [99] Pepper mottle virus PepMoV pPepMoV-I: GFP (with intron 2 of ST-LS1) contains a gfp gene inserted between P1 and HC-Pro The consistent enhancement of PepMoV RNA level and translation products (GFP) observed in the study suggested a hypothesis that the intron ST-LS1 enhanced the stability and translational efficiency of the PepMoV transcripts in the infiltrated leaves. [100] Plum pox virus PPV-NAT pPPV-H6K1-NAT contains a histidine tag inserted in the protein of a 6 kDa (6K1) coding region For detection of 6K1 as a mature protein of 6 kDa in vivo, pPPV-H6K1-NAT enabled the concentration and purification of histidine-tagged 6K1 from infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) through affinity chromatography. [101] Plum pox virus PPV-Rec pIC-PPV-Rec-P1His contains a sequence coding for six histidine residues inserted between the 4th and 5th amino acid of the P1 protein The pIC-PPV-Rec-P1His was able to replicate in N. benthamiana and remained stable during several mechanical passages of the virus. Immunoblot analysis with the anti-his antibody showed a diffuse band corresponding to the molecular weight about 70-80 kDa in the root samples from early stage of infection. However, this signal culminated on the sixth day post inoculation, later it rapidly disappeared. [102] Potato virus Y PVY-RB pGPVY-Ros1 contains a Antirrhinum majus Rosea1 transcription factor inserted between the NIb and CP cistrons • Mechanically inoculated solanaceous plants induced the formation of red infection foci in inoculated tissue and solid dark red pigmentation in systemically infected tissue, which allows disease progression to be easily monitored • Facilitated the novel quantitative analysis of antiviral activity in plants by using silver nanoparticles, a nanomaterial with exciting antimicrobial properties • Enabled the visual monitoring the virus transmission by an aphid vector [94]

Conclusions
Reverse genetics in virology relies on cDNA intermediates in order to genetically manipulate RNA viruses and further produce biologically active RNA molecules. Likewise, the available infectious full-length cDNA clones of potyviruses enable countless reverse genetics studies on phenotypic alteration and cross-protection by mutated viruses as well as in determining viral elements responsible for a particular biological characteristic of the virus. Apart from improving our understanding on the complexities of interactions between host and viral factors, reverse genetics strategies have also made various applications of recombinant vectors possible. Furthermore, the approach contributed immeasurably to the elucidation of basic functions of potyviral proteins, certain motifs or genome sequences in viral replication, transmission, and cell-to-cell movement. All these findings, in conjunction with different approaches such as transciptomics and proteomics analyses, would lead to the building of a larger network that helps to further explore potyviruses, especially in the identification of more durable resistance genes. Hence, reverse genetics technologies of potyviruses are believed to hold great promise for commercial applications in the future. Despite these breakthroughs, although various viral determinants have been identified through plant-potyvirus pathosystems, the host targets of those determinants are yet to be characterized in the future.

Conflicts of Interest:
The authors declare no conflict of interest.