Expanding Possibilities for Foreign Gene Expression by Cucumber Green Mottle Mosaic Virus Genome-Based Bipartite Vector System.

Expanding possibilities for foreign gene expression in cucurbits, we present a novel approach utilising a bipartite vector system based on the cucumber green mottle mosaic virus (CGMMV) genome. Traditional full-length CGMMV vectors face limitations such as a restricted cargo capacity and unstable foreign gene expression. To address these challenges, we developed two 'deconstructed' CGMMV genomes, DG-1 and DG-2. DG-1 features a major internal deletion, resulting in the loss of crucial replicase enzyme domains, rendering it incapable of self-replication. However, a staggered infiltration of DG-1 in CGMMV-infected plants enabled successful replication and movement, facilitating gene-silencing experiments. Conversely, DG-2 was engineered to enhance replication rates and provide multiple cloning sites. Although it exhibited higher replication rates, DG-2 remained localised within infiltrated tissue, displaying trans-replication and restricted movement. Notably, DG-2 demonstrated utility in expressing GFP, with a peak expression observed between 6 and 10 days post-infiltration. Overall, our bipartite system represents a significant advancement in functional genomics, offering a robust tool for foreign gene expression in Nicotiana benthamiana.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Size variations of mesoporous silica nanoparticle control uptake efficiency and delivery of AC2-derived dsRNA for protection against tomato leaf curl New Delhi virus.

KEY MESSAGE: We report the size dependent uptake of dsRNA loaded MSNPs into the leaves and roots of Nicotiana benthamiana plants and accessed for their relative reduction in Tomato leaf curl New Delhi viral load. A non-GMO method of RNA interference (RNAi) has been recently in practice through direct delivery of double stranded RNA into the plant cells. Tomato leaf curl New Delhi virus (ToLCNDV), a bipartitie begomovirus, is a significant viral pathogen of many crops in the Indian subcontinent. Conventional RNAi cargo delivery strategies for instance uses viral vectors and Agrobacterium-facilitated delivery, exhibiting specific host responses from the plant system. In the present study, we synthesized three different sizes of amine-functionalized mesoporous silica nanoparticles (amino-MSNPs) to mediate the delivery of dsRNA derived from the AC2 (dsAC2) gene of ToLCNDV and showed that these dsRNA loaded nanoparticles enabled effective reduction in viral load. Furthermore, we demonstrate that amino-MSNPs protected the dsRNA molecules from nuclease degradation, while the complex was efficiently taken up by the leaves and roots of Nicotiana benthamiana. The real time gene expression evaluation showed that plants treated with nanoparticles of different sizes ~ 10 nm (MSNPDEA), ~ 32 nm (MSNPTEA) and ~ 66 nm (MSNPNH3) showed five-, eleven- and threefold reduction of ToLCNDV in N. benthamiana, respectively compared to the plants treated with naked dsRNA. This work clearly demonstrates the size dependent internalization of amino-MSNPs and relative efficacy in transporting dsRNA into the plant system, which will be useful in convenient topical treatment to protect plants against their pathogens including viruses. Mesoporous silica nanoparticles loaded with FITC, checked for its uptake into Nicotiana benthamiana.

Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression.

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9-16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14-16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Sub-cellular localization of suppressor proteins of tomato leaf curl New Delhi virus.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, is the most important among the 14 species of begomoviruses infecting tomato in Indian subcontinent. Begomovirus is known to  evade RNA silencing of host plants through suppressor proteins. However, in case of ToLCNDV, the suppressor proteins have not been studied well. The objective of the study is to know the sub-cellular localization of three suppressor proteins encoded by AV2, AC2 and AC4 ORFs of ToLCNDV in Nicotiana benthamiana. AV2, AC2 and AC4 ORFs of ToLCNDV were  cloned and sequenced (accession numbers MW423574, MW423576, MW423575, respectively) from a ToLCNDV isolate characterized earlier (accession number MW429271) and GFP tagged constructs were prepared in a plant expressing binary vector pEarleygate103. Bioinformatics analysis using Peptide 2.0 server predicted that all these proteins have more basic amino acid residues then acidic amino acid and AV2 protein has more hydrophobic amino acid residues. ScanProsite server predicted presence of different fuctional motifs in these proteins amongst which presence of kinase motif was observed in all of them. Virus mPLoc server predicted their subcellular localization. The suppressor gene constructs were agroinfiltrated on to leaves of one month old N. benthamiana plants and their subcellular localization has been studied through confocal microscopy. Results have shown that AV2 localizes in the host cell membrane and nucleus, AC2 in the nucleus and AC4 in the host cell membrane. Earlier reports with other begomoviruses also showed similar localization behaviour of these suppressor protein except AV2, where it was shown to be present in cytoplasm. Such localization study will help understand the mechanism of their suppression activity.

Identification and Functional Analysis of Four RNA Silencing Suppressors in Begomovirus Croton Yellow Vein Mosaic Virus.

Croton yellow vein mosaic virus (CYVMV), a species in the genus Begomovirus, is a prolific monopartite begomovirus in the Indian sub-continent. CYVMV infects multiple crop plants to cause leaf curl disease. Plants have developed host RNA silencing mechanisms to defend the threat of viruses, including CYVMV. We characterized four RNA silencing suppressors, namely, V2, C2, and C4 encoded by CYVMV and betasatellite-encoded C1 protein (ßC1) encoded by the cognate betasatellite, croton yellow vein betasatellite (CroYVMB). Their silencing suppressor functions were verified by the ability of restoring the ß-glucuronidase (GUS) activity suppressed by RNA silencing. We showed here for the first time that V2 was capable of self-interacting, as well as interacting with the V1 protein, and could be translocalized to the plasmodesmata in the presence of CYVMV. The knockout of either V2 or V1 impaired the intercellular mobility of CYVMV, indicating their novel coordinated roles in the cell-to-cell movement of the virus. As pathogenicity determinants, each of V2, C2, and C4 could induce typical leaf curl symptoms in Nicotiana benthamiana plants even under transient expression. Interestingly, the transcripts and proteins of all four suppressors could be detected in the systemically infected leaves with no correlation to symptom induction. Overall, our work identifies four silencing suppressors encoded by CYVMV and its cognate betasatellite and reveals their subcellular localizations, interaction behavior, and roles in symptom induction and intercellular virus movement.

Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.

Artificially induced phased siRNAs promote virus resistance in transgenic plants.

We previously developed transgenic tobacco plants that were resistant to two geminiviruses. We generated resistance using RNAi constructs that produced trans-acting siRNA (tasiRNA) like secondary siRNAs known as phased siRNA (phasiRNA) that targeted several regions of Tomato Leaf Curl New Delhi Virus (ToLCNDV) and Tomato Leaf Curl Gujarat Virus (ToLCGV) transcripts encoding the RNA silencing suppressor proteins AC2 and AC4. Here, we performed degradome analysis to determine the precise cleavage sites of RNA-RNA interaction between phasiRNA and viral transcripts. We then applied our RNAi technology in tomato, which is the natural host for ToLCNDV and ToLCGV. The relative ease of developing and using phasiRNA constructs represents a significant technical advance in imparting virus resistance in crops and/or important model systems.

AC4 protein of tomato leaf curl Palampur virus is an RNA silencing suppressor and a pathogenicity determinant.

Plants deploy RNA silencing as a natural defence against invading viruses involving sequence-specific degradation of the viral RNAs. As a counter-defence strategy, viruses encode suppressor proteins that simultaneously target different steps of the silencing machinery. Tomato leaf curl Palampur virus (ToLCPalV) is a bipartite begomovirus in Geminiviridae family. It is responsible for significant reduction in the crop yield and quality. DNA-A of the virus encodes for six proteins whereas DNA-B codes for two proteins. In this study, all viral genes were screened for their role in suppression of green fluorescent protein (GFP) silencing in Nicotiana tabacum cv. Xanthi, employing agrobacterium based co-infiltration assay. The assay identified AC4 as a potential suppressor of RNA silencing. In addition, AC4 expression also suppressed virus-induced gene silencing (VIGS) of the phytoene desaturase (PDS) gene in N. benthamiana. Potato virus X (PVX) mediated transient expression of the AC4 in N. benthamiana showed enhanced symptoms that include downward leaf curling, leaf puckering and tissue necrosis. Further, N. benthamiana lines stably expressing AC4 showed severe developmental abnormalities. Mutational analysis suggested that glycine at 2nd position is essential for AC4 pathogenicity. Collectively, these findings demonstrate the role of ToLCPalV AC4 in viral pathogenesis, disease establishment and suppression of gene silencing.

Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology.

The success of Bt transgenics in controlling predation of crops has been tempered by sporadic emergence of resistance in targeted insect larvae. Such emerging threats have prompted the search for novel insecticidal molecules that are specific and could be expressed through plants. We have resorted to small RNA-based technology for an investigative search and focused our attention to an insect-specific miRNA that interferes with the insect molting process resulting in the death of the larvae. In this study, we report the designing of a vector that produces artificial microRNA (amiR), namely amiR-24, which targets the chitinase gene of Helicoverpa armigera. This vector was used as transgene in tobacco. Northern blot and real-time analysis revealed the high level expression of amiR-24 in transgenic tobacco plants. Larvae feeding on the transgenic plants ceased to molt further and eventually died. Our results demonstrate that transgenic tobacco plants can express amiR-24 insectice specific to H. armigera.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, groundnut bud necrosis virus.

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Development of plants resistant to tomato geminiviruses using artificial trans-acting small interfering RNA.

RNA interference (RNAi), a conserved RNA-mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter-strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down-regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans-acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single-step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro-infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro-infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA-generating constructs, and T1 plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.

Key elements of the RNAi pathway are regulated by hepatitis B virus replication and HBx acts as a viral suppressor of RNA silencing.

The host-mediated RNAi pathways restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show in the present study that the siRNA-mediated silencing of Drosha, Dicer and Ago2 [argonaute RISC (RNA-induced silencing complex) catalytic component 2] transcripts in Huh7 cells resulted in elevated levels of HBV (hepatitis B virus)-specific RNAs and, conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in CHB (chronic hepatitis B) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RSS (RNA-silencing suppressor) properties. Using GFP-based reversion of silencing assays, in the present study we found that HBx is an RSS protein. Through a series of deletions and substitution mutants, we found that the full-length HBx protein is required for optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer-mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defence, in particular by abrogating the function of Dicer. The present study may have implications in the development of newer strategies to combat HBV infection.

Unfolding of in planta activity of anti-rep ribozyme in presence of a RNA silencing suppressor.

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double-stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep-mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA-silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA-silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down-regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.

MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.

Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase.

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).