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.

Genomic properties of allamanda leaf mottle distortion virus, a new begomovirus from golden trumpet (Allamanda cathartica) in India.

Golden trumpet (Allamanda cathartica) plants were observed to exhibit mottling and distortion symptoms on leaves. The genome of an associated begomovirus (Al-K1) was amplified by rolling-circle amplification, cloned, and sequenced. The viral genome consisted of two circular ssDNA molecules, and the organization of the ORFs was similar to those of DNA-A and DNA-B components of bipartite begomoviruses. The size of DNA-A (KC202818) and DNA-B (MG969497) of the begomovirus was 2772 and 2690 nucleotides, respectively. Sequence analysis revealed that the DNA-A and DNA-B components shared the highest sequence identity with duranta leaf curl virus (MN537564, 87.8%) and cotton leaf curl Alabad virus (MH760452, 81.0%), respectively. Interestingly, the Al-K1 isolate shared significantly less nucleotide sequence identity with allamanda leaf curl virus (EF602306, 71.6%), the only monopartite begomovirus reported previously in golden trumpet from China. Al-K1 shared less than 91% sequence identity with other begomoviruses, and hence, according to the latest ICTV guidelines for species demarcation of begomoviruses, Al-K1 is proposed to be a member of a new species, and we propose the name "allamanda leaf mottle distortion virus" (AllLMoDV-[IN-Al_K1-12]) for this virus. AllLMoDV was detected in various golden trumpet samples from different locations by PCR with specific primers based on the genome sequence determined in this study. Our study provides evidence of the occurrence of a new bipartite begomovirus in a perennial ornamental plant in India.

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.

3' and 5' microRNA-end post-biogenesis modifications in plant transcriptomes: Evidences from small RNA next generation sequencing data analysis.

The processing of miRNA from its precursors is a precisely regulated process and after biogenesis, the miRNAs are amenable to different kinds of modifications by the addition or deletion of nucleotides at the terminal ends. However, the mechanism and functions of such modifications are not well studied in plants. In this study, we have specifically analysed the terminal end non-templated miRNA modifications, using NGS data of rice, tomato and Arabidopsis small RNA transcriptomes from different tissues and physiological conditions. Our analysis reveals template independent terminal end modifications in the mature as well as passenger strands of the miRNA duplex. Interestingly, it is also observed that miRNA sequences terminating with a cytosine (C) at the 3' end undergo a higher percentage of 5' end modifications. The terminal end modifications did not correlate with the miRNA abundances and are independent of tissue types, physiological conditions and plant species. Our analysis indicates that the addition of nucleotides at miRNA ends is not influenced by the absence of RNA dependent RNA polymerase 6. Moreover the terminal end modified miRNAs are also observed amongst AGO1 bound small RNAs and have potential to alter target, indicating its important functional role in repression of gene expression.

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.

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.

Arabidopsis thaliana NAC083 protein interacts with Mungbean yellow mosaic India virus (MYMIV) Rep protein.

Geminiviral replication initiator protein (Rep) is a key player in geminiviral rolling circle mode of replication. However, the virus exploits various host cellular machineries for its replication. Study of these host factors is important to understand the geminiviral DNA replication in greater details. With this view, we screened for the peptides interacting with the Rep protein of a representative of geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), employing phage display technique. Through this screen, we have identified a host transcription factor, NAC083, as a potential MYMIV-Rep-binding partner. In silico docking studies also suggested possible binding of NAC083 peptide to MYMIV-Rep. We validated the interaction between MYMIV-Rep and Arabidopsis thaliana full-length NAC083 protein using in vitro pull-down assay and yeast two-hybrid analysis. NAC proteins are well-known transcription factors belonging to the largest gene families in plants. This study demonstrates for the first time the interaction of NAC083, a member of NAC transcription factor family, with MYMIV-Rep protein thereby indicating its possible role in MYMIV DNA replication.

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.

A novel role for RAD54: this host protein modulates geminiviral DNA replication.

Geminiviruses primarily encode only few factors, such as replication initiator protein (Rep), and need various host cellular machineries for rolling-circle replication (RCR) and/or recombination-dependent replication (RDR). We have identified a host factor, RAD54, in a screen for Rep-interacting partners and observed its role in DNA replication of the geminivirus mungbean yellow mosaic India virus (MYMIV). We identified the interacting domains ScRAD54 and MYMIV-Rep and observed that ScRAD54 enhanced MYMIV-Rep nicking, ATPase, and helicase activities. An in vitro replication assay demonstrated that the geminiviral DNA replication reaction depends on the viral Rep protein, viral origin of replication sequences, and host cell-cycle proteins. Rad54-deficient yeast nuclear extract did not support in vitro viral DNA replication, while exogenous addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in in planta replication was confirmed by the transient replication assay; i.e., agroinoculation studies. RAD54 is a well-known recombination/repair protein that uses its DNA-dependent ATPase activity in conjunction with several other host factors. However, this study demonstrates for the first time that the eukaryotic rolling-circle replicon depends on the RAD54 protein.

Identification of mirtrons in rice using MirtronPred: a tool for predicting plant mirtrons.

Studies from flies and insects have reported the existence of a special class of miRNA, called mirtrons that are produced from spliced-out introns in a DROSHA-independent manner. The spliced-out lariat is debranched and refolded into a stem-loop structure resembling the pre-miRNA, which can then be processed by DICER into mature ~21 nt species. The mirtrons have not been reported from plants. In this study, we present MirtronPred, a web based server to predict mirtrons from intronic sequences. We have used the server to predict 70 mirtrons in rice introns that were put through a stringent selection filter to shortlist 16 best sequences. The prediction accuracy was subsequently validated by northern analysis and RT-PCR of a predicted Os-mirtron-109. The target sequences for this mirtron were also found in the rice degradome database. The possible role of the mirtron in rice regulon is discussed. The MirtronPred web server is available at http://bioinfo.icgeb.res.in/mirtronPred.

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.

A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

Severe symptoms of cotton leaf curl disease (CLCuD) are caused by the association of a single-stranded circular DNA satellite (betasatellite) with a helper begomovirus. In this study, we analyzed 40 leaf samples (primarily cotton with CLCuD symptoms and other plants growing close by) from four sites between New Delhi and the Pakistan/India border, using rolling-circle amplification (RCA) and PCR. In total, the complete sequences of 12 different helper viruses, eight alphasatellites, and one betasatellite from five different plant species were obtained. A recombinant helper virus molecule found in okra and a novel alphasatellite-related DNA from croton are also described. This is the first report of the presence of both DNA components (helper virus and betasatellite) associated with resistance-breaking CLCuD in India, and it highlights the need for further work to combat its damage and spread.

Silencing of Thrips palmi UHRF1BP1 and PFAS Using Antisense Oligos Induces Mortality and Reduces Tospovirus Titer in Its Vector

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, legumes, and ornamentals. In addition, it transmits several plant viruses. T. palmi genes associated with innate immunity, endocytosis-related pathways, and cuticular development are highly enriched in response to Groundnut bud necrosis orthotospovirus (GBNV, genus Orthotospovirus, family Tospoviridae) infection. As the previous transcriptomic study suggested the involvement of T. palmi UHRF1BP1 and PFAS in GBNV infection, these two genes were targeted for silencing using antisense oligonucleotides (ASOs), and the effects on thrips' fitness and virus acquisition were observed. Phosphorothioate modification of ASOs was carried out by replacing the nonbridging oxygen atom with a sulfur atom at the 3' position to increase nuclease stability. The modified ASOs were delivered orally through an artificial diet. Exposure to ASOs reduced the target mRNA expression up to 2.70-fold optimally. Silencing of T. palmi UHRF1BP1 and PFAS induced 93.33% mortality that further increased up to 100% with an increase in exposure. Silencing of T. palmi UHRF1BP1 and PFAS also produced morphological deformities in the treated T. palmi. GBNV titer in T. palmi significantly declined post-exposure to ASOs. This is the first-ever report of silencing T. palmi UHRF1BP1 and PFAS using modified ASO to induce mortality and impair virus transmission in T. palmi. T. palmi UHRF1BP1 and PFAS would be novel genetic targets to manage thrips and restrict the spread of tospovirus.

A Method for Developing RNAi-Derived Resistance in Cowpea Against Geminiviruses.

In plants, RNA interference (RNAi) is triggered by double-stranded RNA (dsRNA). Accordingly, various RNA silencing technologies involving hpRNA, artificial microRNA (miRNA), and virus-induced gene silencing (VIGS) are used for controlling the expression of genes. Such manipulations help understanding gene functions and crop improvement biotechnology. A typical hpRNA construct is comprised of an intron splicable perfect inverted repeat of the target gene sequences under the control of a strong promoter. Geminiviruses, especially Mungbean Yellow Mosaic India Virus (MYMIV) cause devastating diseases in legume plants including cowpea, incurring severe crop loss. RNAi, involving hpRNA construct as transgene, is used to control these diseases at the early stages of geminivirus infection in the host, preventing symptom development and viral DNA accumulation. In this chapter, we describe a detailed protocol for the identification of geminivirus isolates from the filed grown cowpea plants, characterization of virus isolates under the laboratory conditions, design and construct RNAi vectors for effective suppression of viral target genes, and consequent development of transgenic cowpea using Agrobacterium-mediated transformation protocol. These transgenics are subsequently evaluated for resistance to MYMIV.

Random distribution of nucleotide polymorphism throughout the genome of tomato-infecting begomovirus species occurring in India: implication in PCR based diagnosis.

Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00785-9.

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.

Transovarial Transmission of Dolichos Yellow Mosaic Virus by Its Vector, Bemisia tabaci Asia II 1.

The cultivation of dolichos bean [Lablab purpureus (L.) Sweet] has been severely affected by dolichos yellow mosaic virus (DoYMV, Begomovirus) transmitted by whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). We tested the transovarial transmission of DoYMV in next-generation B. tabaci by PCR, real-time PCR, Southern blot hybridization, and biological transmission. The eggs, laid by DoYMV-exposed B. tabaci, carry the virus in a unique pattern. Only the eggs laid in between 3 and 6 days post virus acquisition by a parent B. tabaci were DoYMV positive. When tested individually in real-time PCR, around 31-53% of the eggs carried the virus. The presence of DoYMV in ovaries and F1 eggs was further substantiated by the hybridization of a Cy3-conjugated nucleic acid probe complementary to the viral strand of DoYMV. Viral DNA was also detected in F1 adults and F2 eggs. B. tabaci progenies carried not only the DoYMV DNA but were also infective. The F1 adults transmitted DoYMV to all tested plants and produced strong yellow mosaic symptoms. An increase in viral copies from egg to nymphal stage indicated propagation of DoYMV in B. tabaci. However, the increase was for a short period and decreased thereafter. The present study provides the first evidence of transovarial transmission and propagation of a bipartite begomovirus in its vector, B. tabaci Asia II 1. The transovarial transmission and replication of DoYMV in B. tabaci have great epidemiological relevance as B. tabaci can serve as a major host of the virus to bridge the gap between the cropping seasons.

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.

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.