An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication.

Persistent or chronic infection with the hepatitis B virus (HBV) represents one of the most common viral diseases in humans. The hepatitis B virus deploys the hepatitis B virus X protein (HBx) as a suppressor of host defenses consisting of RNAi-based silencing of viral genes. Because of its critical role in countering host defenses, HBx represents an attractive target for antiviral drugs. Here, we developed and optimized a loss-of-function screening procedure, which identified a potential pharmacophore that abrogated HBx RNAi suppression activity. In a survey of 14,400 compounds in the Maybridge Screening Collection, we prioritized candidate compounds via high-throughput screening based on reversal of green fluorescent protein (GFP)-reported, RNAi-mediated silencing in a HepG2/GFP-shRNA RNAi sensor line. The screening yielded a pharmacologically active compound, N-(2,4-difluorophenyl)-N'-[3-(1H-imidazol-1-yl) propyl] thiourea (IR415), which blocked HBx-mediated RNAi suppression indicated by the GFP reporter assay. We also found that IR415 reversed the inhibitory effect of HBx protein on activity of the Dicer endoribonuclease. We further confirmed the results of the primary screen in IR415-treated, HBV-infected HepG2 cells, which exhibited a marked depletion of HBV core protein synthesis and down-regulation of pre-genomic HBV RNA. Using a molecular interaction analysis system, we confirmed that IR415 selectively targets HBx in a concentration-dependent manner. The screening assay presented here allows rapid and improved detection of small-molecule inhibitors of HBx and related viral proteins. The assay may therefore potentiate the development of next-generation RNAi pathway-based therapeutics and promises to accelerate our search for novel and effective drugs in antiviral research.

s8ORF2 protein of infectious salmon anaemia virus is a RNA-silencing suppressor and interacts with Salmon salar Mov10 (SsMov10) of the host RNAi machinery.

The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections.

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.

Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner.

RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication.

Association of HADHA with human RNA silencing machinery.

In RNA silencing, small RNAs produced by dicer mediate target repression guided by RNA induced silencing complex (RISC). To effectively mediate this response, one or more proteins are employed at each stage. In the present study, we investigated HADHA, a new component in the human RNA silencing machinery. Immunoprecipitation analysis revealed that, HADHA associates with dicer complex and immunohistochemical studies confirmed its co localization with Dicer in the cytoplasm. Further, over expression of HADHA resulted in higher abundance levels of mature miRNA against a reduction in respective precursor levels and vice versa in HADHA knocked down cells. These findings suggest an auxiliary role for HADHA in miRNA biogenesis and help in better understanding of molecular mechanisms underlying RNAi pathway in mammals.

A draft genome assembly of the army worm, Spodoptera frugiperda.

Spodoptera is an agriculturally important pest insect and studies in understanding its biology have been limited by the unavailability of its genome. In the present study, the genomic DNA was sequenced and assembled into 37,243 scaffolds of size, 358 Mb with N50 of 53.7 kb. Based on degree of identity, we could anchor 305 Mb of the genome onto all the 28 chromosomes of Bombyx mori. Repeat elements were identified, which accounts for 20.28% of the total genome. Further, we predicted 11,595 genes, with an average intron length of 726 bp. The genes were annotated and domain analysis revealed that Sf genes share a significant homology and expression pattern with B. mori, despite differences in KOG gene categories and representation of certain protein families. The present study on Sf genome would help in the characterization of cellular pathways to understand its biology and comparative evolutionary studies among lepidopteran family members to help annotate their genomes.

Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins.

BACKGROUND: RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application. RESULTS: The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects. CONCLUSION: The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.

Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor.

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication.

Identification of tissue-preferential expression patterns of rice miRNAs.

It is imperative to understand the mechanisms of growth and development in higher plants for improving plant adaptation during different developmental stages. Plant microRNAs (miRs) play crucial regulatory roles in various developmental processes. As many as 15 miR families having multiple members are known to regulate plant development, yet the spatio-temporal expression patterns of individual members are not fully characterized. It is likely that different members of miR families can make specific contributions to the spatio-temporal control of targets. To understand the functional complexity of miRs and the amount of degeneracy existing in miR-mediated regulation of differentiated but developing tissues, we have identified the Osa-miR-sequences that are expressed in specific tissues. We adopted the approach of comparative miR profiling using next-generation sequencing technology followed by experimental validation. It was observed that 59 Osa-miR-sequences show tissue-preferential expression in local basmati rice variety; while 126 miRs belonging to 81 families are differentially regulated in these tissues. The 21 nt miRs were predominant in all tissues, but the 24 nt miRs were the most abundantly expressed. This indicates that target cleavage and chromatin state regulation are involved in organ development. This study also identified the expression patterns of individual members of Osa-miR families that were common and divergent between the indica and japonica rice varieties. The expression patterns of the predicted targets were also analyzed. The possible implications of the miR distribution patterns with respect to the regulation of their respective targets are discussed.

The host factor RAD51 is involved in mungbean yellow mosaic India virus (MYMIV) DNA replication.

Geminiviruses replicate their single-stranded genomes with the help of only a few viral factors and various host cellular proteins primarily by rolling-circle replication (RCR) and/or recombination-dependent replication. AtRAD51 has been identified, using the phage display technique, as a host factor that potentially interacts with the Rep protein of mungbean yellow mosaic India virus (MYMIV), a member of the genus Begomovirus. In this study, we demonstrate the interaction between MYMIV Rep and a host factor, AtRAD51, using yeast two-hybrid and ß-galactosidase assays, and this interaction was confirmed using a co-immunoprecipitation assay. The AtRAD51 protein complemented the rad51∆ mutation of Saccharomyces cerevisiae in an ex vivo yeast-based geminivirus DNA replication restoration assay. The semiquantitative RT-PCR and northern hybridization data revealed a higher level of expression of the Rad51 transcript in MYMIV-infected mungbean than in uninfected, healthy plants. Our findings provide evidence for a possible cross-talk between RAD51 and MYMIV Rep, which essentially controls viral DNA replication in plants, presumably in conjunction with other host factors. The present study demonstrates for the first time the involvement of a eukaryotic RAD51 protein in MYMIV replication, and this is expected to shed light on the machinery involved in begomovirus DNA replication.

Arabidopsis thaliana MCM2 plays role(s) in mungbean yellow mosaic India virus (MYMIV) DNA replication.

Geminiviruses are plant pathogens with single-stranded (ss) DNA genomes of about 2.7 kb in size. They replicate primarily via rolling-circle replication (RCR) with the help of a few virally encoded factors and various host-cell machineries. The virally encoded replication initiator protein (Rep) is essential for geminivirus replication. In this study, by interaction screening of an Arabidopsis thaliana cDNA library, we have identified a host factor, MCM2, that interacts with the Rep protein of the geminivirus mungbean yellow mosaic India virus (MYMIV). Using yeast two-hybrid, ß-galactosidase and co-immunoprecipitation assays, we demonstrated an interaction between MYMIV-Rep and the host factor AtMCM2. We investigated the possible role of AtMCM2 in geminiviral replication using a yeast-based geminivirus DNA replication restoration assay and observed that the AtMCM2 protein complemented the mcm2∆ mutation of S. cerevisiae. Our data suggest the involvement of AtMCM2 in the replication of MYMIV ex vivo. The role of MCM2 in replication was confirmed in planta by a transient replication assay in both wild-type and mutant Arabidopsis plants through agroinoculation. Our data provide evidence for the involvement of AtMCM2 in geminiviral DNA replication, presumably in conjunction with other host factors, and suggest its importance in MYMIV DNA replication.

Micro-regulators of auxin action.

microRNAs (miRs) are 21- to 24-nucleotide-long RNA molecules that are mainly involved in regulating the gene expression at the post-transcriptional levels. They are present in a variety of organisms from algae to plants and play an important role in gene regulation. The identification of several diverging and converging functions of miRs indicates that they play versatile roles in regulating plant development including differentiation, organ development, phase change, signalling, disease resistance and response to environmental stresses. This article provides a concise update on the plant miR functions and their targets in the auxin pathway with focus on the interactions between miRs and auxin signalling to intricately regulate the plant responses.

Host microRNAs exhibit differential propensity to interact with SARS-CoV-2 and variants of concern.

A significant number of SARS-CoV-2-infected individuals naturally overcome viral infection, suggesting the existence of a potent endogenous antiviral mechanism. As an innate defense mechanism, microRNA (miRNA) pathways in mammals have evolved to restrict viruses, besides regulating endogenous mRNAs. In this study, we systematically examined the complete repertoire of human miRNAs for potential binding sites on SARS-CoV-2 Wuhan-Hu-1, Beta, Delta, and Omicron. Human miRNA and viral genome interaction were analyzed using RNAhybrid 2.2 with stringent parameters to identify highly bonafide miRNA targets. Using publicly available data, we filtered for miRNAs expressed in lung epithelial cells/tissue and oral keratinocytes, concentrating on the miRNAs that target SARS-CoV-2 S protein mRNAs. Our results show a significant loss of human miRNA and SARS-CoV-2 interactions in Omicron (130 miRNAs) compared to Wuhan-Hu-1 (271 miRNAs), Beta (279 miRNAs), and Delta (275 miRNAs). In particular, hsa-miR-3150b-3p and hsa-miR-4784 show binding affinity for S protein of Wuhan strain but not Beta, Delta, and Omicron. Loss of miRNA binding sites on N protein was also observed for Omicron. Through Ingenuity Pathway Analysis (IPA), we examined the experimentally validated and highly predicted functional role of these miRNAs. We found that hsa-miR-3150b-3p and hsa-miR-4784 have several experimentally validated or highly predicted target genes in the Toll-like receptor, IL-17, Th1, Th2, interferon, and coronavirus pathogenesis pathways. Focusing on the coronavirus pathogenesis pathway, we found that hsa-miR-3150b-3p and hsa-miR-4784 are highly predicted to target MAPK13. Exploring miRNAs to manipulate viral genome/gene expression can provide a promising strategy with successful outcomes by targeting specific VOCs.

The 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an RNA silencing suppressor.

RNA silencing suppressors (RSSs) are well studied for plant viruses but are not well defined to date for animal viruses. Here, we have identified an RSS from a medically important positive-sense mammalian virus, Severe acute respiratory syndrome coronavirus. The viral 7a accessory protein suppressed both transgene and virus-induced gene silencing by reducing the levels of small interfering RNA (siRNA). The suppression of silencing was analyzed by two independent assays, and the middle region (amino acids [aa] 32 to 89) of 7a was responsible for suppression. Finally, the RNA suppression property and the enhancement of heterologous replicon activity by the 7a protein were confirmed for animal cell lines.

Molecular characterization and infectivity of a Tomato leaf curl New Delhi virus variant associated with newly emerging yellow mosaic disease of eggplant in India.

BACKGROUND: Begomoviruses have emerged as serious problem for vegetable and fiber crops in the recent past, frequently in tropical and subtropical region of the world. The association of begomovirus with eggplant yellow mosaic disease is hitherto unknown apart from one report from Thailand. A survey in Nagpur, Central India, in 2009-2010 showed severe incidence of eggplant yellow mosaic disease. Here, we have identified and characterized a begomovirus responsible for the newly emerging yellow mosaic disease of eggplant in India. RESULTS: The complete DNA-A and DNA-B genomic components of the causative virus were cloned and sequenced. Nucleotide sequence analysis of DNA-A showed that it shared highest 97.6% identity with Tomato leaf curl New Delhi virus-India[India:Udaipur:Okra:2007] and lowest 87.9% identity with Tomato leaf curl New Delhi virus-India[India:NewDelhi:Papaya:2005], while DNA-B showed highest 94.1% identity with ToLCNDV-IN[IN:UD:Ok:07] and lowest 76.2% identity with ToLCNDV-India[India:Lucknow]. Thus, it appears that this begomovirus is a variant of ubiquitous ToLCNDV and hence, we suggest the name ToLCNDV-India[India:Nagpur:Eggplant:2009] for this variant. The pathogenicity of ToLCNDV-IN[IN:Nag:Egg:09] isolate was confirmed by agroinfiltraion and dimeric clones of DNA-A and DNA-B induced characteristic yellow mosaic symptoms in eggplants and leaf curling in tomato plants. CONCLUSION: This is the first report of a ToLCNDV variant moving to a new agriculturally important host, eggplant and causing yellow mosaic disease. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with eggplant yellow mosaic disease.

The presence of tomato leaf curl Kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants.

BACKGROUND: Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail. RESULTS: We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants. CONCLUSIONS: Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein.

Overexpression of an insect virus encoded silencing suppressor does not enhance plants' susceptibility to its natural virus.

RNA silencing plays a key role in shielding plant and animal hosts against viral invasion and infection. Viruses encode RNA silencing suppressors (RSS) to block small RNA guided silencing of viral transcripts. The B2 protein encoded by Flock House virus (FHV) is a well-characterized RSS that facilitates infection in insects. It has been shown to act as a functional RSS in plants. FHVB2 over-expressing tobacco plants were used to study the effect of RSS on plant susceptibility to Tobacco mosaic virus (TMV), its natural pathogen. The major symptoms observed in TMV-infected transgenic plants were greenish mosaic, puckering and distortion of leaves, but the infected transgenic leaves were able to resist chlorophyll loss. The infected leaves of transgenic plants showed no significant difference in accumulation of virus when compared with that of the wild type plants. FHVB2 plants showed higher levels of H2O2 and the ROS scavenging enzymes, APX and SOD. This suggests that interference of FHVB2 with RNA silencing machinery may activate alternative defense pathways in the plants so that they are not overly sensitive to TMV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-020-00644-5.

Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology.

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and trans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant-virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1).

BACKGROUND: Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study. RESULTS: AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro. CONCLUSIONS: Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

MicroRNA profiling of tomato leaf curl New Delhi virus (tolcndv) infected tomato leaves indicates that deregulation of mir159/319 and mir172 might be linked with leaf curl disease.

BACKGROUND: Tomato leaf curl virus (ToLCV), a constituent of the genus Begomovirus, infects tomato and other plants with a hallmark disease symptom of upward leaf curling. Since microRNAs (miRs) are known to control plants developmental processes, we evaluated the roles of miRNAs in Tomato leaf curl New Delhi virus (ToLCNDV) induced leaf curling. RESULTS: Microarray analyses of miRNAs, isolated from the leaves of both healthy and ToLCNDV agroinfected tomato cv Pusa Ruby, revealed that ToLCNDV infection significantly deregulated various miRNAs representing ~13 different conserved families (e.g., miR319, miR172, etc.). The precursors of these miRNAs showed similar deregulated patterns, indicating that the transcription regulation of respective miRNA genes was perhaps the cause of deregulation. The expression levels of the miRNA-targeted genes were antagonistic with respect to the amount of corresponding miRNA. Such deregulation was tissue-specific in nature as no analogous misexpression was found in flowers. The accumulation of miR159/319 and miR172 was observed to increase with the days post inoculation (dpi) of ToLCNDV agroinfection in tomato cv Pusa Ruby. Similarly, these miRs were also induced in ToLCNDV agroinfected tomato cv JK Asha and chilli plants, both exhibiting leaf curl symptoms. Our results indicate that miR159/319 and miR172 might be associated with leaf curl symptoms. This report raises the possibility of using miRNA(s) as potential signature molecules for ToLCNDV infection. CONCLUSIONS: The expression of several host miRNAs is affected in response to viral infection. The levels of the corresponding pre-miRs and the predicted targets were also deregulated. This change in miRNA expression levels was specific to leaf tissues and observed to be associated with disease progression. Thus, certain host miRs are likely indicator of viral infection and could be potentially employed to develop viral resistance strategies.