Capsicum-infecting begomoviruses as global pathogens: host-virus interplay, pathogenesis, and management.

Whitefly-transmitted begomoviruses are among the major threats to the cultivation of Capsicum spp. (Family: Solanaceae) worldwide. Capsicum-infecting begomoviruses (CIBs) have a broad host range and are commonly found in mixed infections, which, in turn, fuels the emergence of better-adapted species through intraspecies and interspecies recombination. Virus-encoded proteins hijack host factors to breach the well-coordinated antiviral response of plants. Epigenetic modifications of histones associated with viral minichromosomes play a critical role in this molecular arms race. Moreover, the association of DNA satellites further enhances the virulence of CIBs as the subviral agents aid the helper viruses to circumvent plant antiviral defense and facilitate expansion of their host range and disease development. The objective of this review is to provide a comprehensive overview on various aspects of CIBs such as their emergence, epidemiology, mechanism of pathogenesis, and the management protocols being employed for combating them.

Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India.

Geminiviridae comprises the largest family of plant viruses which causes severe crop losses in India. The highest pungency chilli Bhut-Jolokia or ghost pepper (Capsicum chinense Jaqc.) hails from North-East region of India and is used in many dishes to add flavors and also for its medicinal value. However, this chilli variety is also affected by viruses leading to crop and economic losses. The present study reports the identification of begomoviruses in the infected chilli Bhut-Jolokia leaf samples collected from eight different places of North-East region (Manipur) of India. The infected leaf samples were screened for the presence of viral genome by rolling circle amplification (RCA) followed by PCR using degenerate primer pairs. The subsequent analyses using restriction fragment length polymorphism and sequencing revealed the presence of Cotton leaf curl Multan virus (CLCuMuV), and Tomato leaf curl Patna betasatellite (ToLCPaB). The findings focus on the phylogenetic relatedness, probable recombinational hot-spots and evolutionary divergence of the viral DNA sequences with the current reported begomoviral genome. To the best of our knowledge, this is the first report showing the presence of CLCuMuV, and associated non-cognate ToLCPaB with leaf curl disease of Bhut-Jolokia chillies. The study reveals potential recombination sites on both viral genome and betsatellite which, during the course of evolution, may have aided the virus to progress and successfully establish infection in chilli plants. Taken together, our results suggest a possible spread of CLCuMuV to the hitherto non-host crop in the North-East region of India.

Host-derived artificial miRNA-mediated silencing of ecdysone receptor gene provides enhanced resistance to Helicoverpa armigera in tomato.

Helicoverpa armigera causes huge crop losses due to its polyphagous nature. The present study demonstrates the use of artificial microRNA (amiRNA) mediated gene silencing approach to generate insect resistant tomato plants. Ecdysone receptor (HaEcR) gene of the target pest, H. armigera, which is involved in the regulation of all developmental stages of the insect life cycle, was silenced by sequence-specific amiRNA (amiRNA-HaEcR). Continuous feeding on detached tomato leaves expressing the amiRNA-319a-HaEcR resulted in reduced target gene transcripts and affected the overall growth and survival of H. armigera. Not only the target gene was down-regulated but, the feeding also affected the expression of down-stream genes involved in the ecdysone signaling pathway. The resistant trait was also observed in T1 generation of tomato transgenic lines. These results further established the role of EcR as a master regulator in insect development and effectiveness of amiRNA technology for efficient control of H. armigera.

Virus and Viroid-Derived Small RNAs as Modulators of Host Gene Expression: Molecular Insights Into Pathogenesis.

Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant's defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.

Co-expression of anti-miR319g and miRStv_11 lead to enhanced steviol glycosides content in Stevia rebaudiana.

BACKGROUND: miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents. RESULTS: The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents. CONCLUSION: In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.

Carotenoid profiling, in silico analysis and transcript profiling of miRNAs targeting carotenoid biosynthetic pathway genes in different developmental tissues of tomato.

Carotenoid biosynthetic pathway is one of the highly significant and very well elucidated secondary metabolic pathways in plants. microRNAs are the potential regulators, widely known for playing a pivotal role in the regulation of various biological as well as metabolic processes. miRNAs may assist in the metabolic engineering of the secondary metabolites for the production of elite genotypes with increased biomass and content of various metabolites. miRNA mediated regulation of carotenoid biosynthetic genes has not been elucidated so far. To illustrate the potential regulatory role of miRNAs in carotenoid biosynthesis, transcript profiling of the known miRNAs and their possible target carotenoid genes was undertaken at eight different developmental stages of tomato, using stem-loop PCR approach combined with quantitative RT-PCR. The inter-relationship amongst carotenoid content, biosynthetic genes and miRNAs was studied in depth. Comparative expression profiles of miRNA and target genes showed variable expression in different tissues studied. The expression level of miRNAs and their target carotenoid genes displayed similar pattern in the vegetative tissues as compared to the reproductive ones, viz. fruit (different stages), indicating the possibility of regulation of carotenoid biosynthesis at various stages of fruit development. This was later confirmed by the HPLC analysis of the carotenoids. The present study has further enhanced the understanding of regulation of carotenoid biosynthetic pathway in plants. The identified miRNAs can be employed to manipulate the biosynthesis of different carotenoids, through metabolic engineering for the production of lycopene rich tomatoes.

Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis in Helicoverpa armigera.

The insect pests are real threat to farmers as they affect the crop yield to a great extent. The use of chemical pesticides for insect pest control has always been a matter of concern as they pollute the environment and are also harmful for human health. Bt (Bacillus thuringensis) technology helped the farmers to get rid of the insect pests, but experienced a major drawback due to the evolution of insects gaining resistance towards these toxins. Hence, alternative strategies are high on demand to control insect pests. RNA-based gene silencing is emerging as a potential tool to tackle with this problem. In this study, we have shown the use of artificial microRNA (amiRNA) to specifically target the ecdysone receptor (EcR) gene of Helicoverpa armigera (cotton bollworm), which attacks several important crops like cotton, tomato chickpea, pigeon pea, etc and causes huge yield losses. Insect let-7a precursor miRNA (pre-miRNA) backbone was used to replace the native miRNA with that of amiRNA. The precursor backbone carrying the 21 nucleotide amiRNA sequence targeting HaEcR was cloned in bacterial L4440 vector for in vitro insect feeding experiments. Larvae fed with Escherichia coli expressing amiRNA-HaEcR showed a reduction in the expression of target gene as well as genes involved in the ecdysone signaling pathway downstream to EcR and exhibited mortality and developmental defects. Stem-loop RT-PCR revealed the presence of amiRNA in the insect larvae after feeding bacteria expressing amiRNA-HaEcR, which was otherwise absent in controls. We also found a significant drop in the reproduction potential (oogenesis) of moths which emerged from treated larvae as compared to control. These results demonstrate the successful use of an insect pre-miRNA backbone to express amiRNA for gene silencing studies in insects. The method is cost effective and can be exploited as an efficient and alternative tool for insect pest management.