Institutional Publications
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Item Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato(Nature Publishing Group, 2016) Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, ManojInvolvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato.Item Post-transcriptional and epigenetic arms of RNA silencing: a defense machinery of naturally tolerant tomato plant against Tomato leaf curl New Delhi virus(Springer, 2014) Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Prasad, ManojTomato leaf curl disease (ToLCD), caused by strains of Tomato leaf curl virus, is major constraint to tomato production globally. The present study was aimed to understand the mechanisms of ToLCD tolerance in a naturally tolerant tomato cultivar through post-transcriptional and DNA methylation-specific RNA silencing. We evaluated the distribution of virus-derived short-interfering RNAs (siRNAs) throughout the Tomato leaf curl New Delhi virus (ToLCNDV) genome along with DNA methylation patterns in intergenic (IR) and Rep (AC1) regions in two tomato cultivars differing in their ToLCNDV tolerance. The methylation pattern was correlated by expression analysis of key methyltransferases genes. In the tolerant cultivar, higher accumulation of viral IR-specific 24-nucleotides (nt) siRNA and AC1-specific 21-nt siRNA were found. Higher methylation levels were observed in various regions of IR. Additionally, AC1 region which facilitates binding of plant nuclear proteins was hypermethylated. DNA methylation in the key regulating region may control the expression of AC1, AC2, and AC3 genes. Components of RNA silencing and DNA methylation machinery were found to be differentially expressed in both the cultivar of tomato at 21 dpi. Thus, we infer that both viral DNA methylation and siRNA-mediated RNA degradation play an important role in conferring tolerance against Tomato leaf curl New Delhi virus. Due to the inability to achieve field resistance in transgenic tomato by deploying the viral genes, targeting the viral genomic regions through RNAi technology reported here could offer an alternate defense strategy for generating transgenics to prevent yield loss.Item Involvement of host regulatory pathways during geminivirus infection: a novel platform for generating durable resistance(Springer, 2014) Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Muthamilarasan, Mehanathan; Prasad, ManojGeminiviruses are widely distributed throughout the world and cause devastating yield losses in almost all the economically important crops. In this review, the newly identified roles of various novel plant factors and pathways participating in plant–virus interaction are summarized with a particular focus on the exploitation of various pathways involving ubiquitin/26S proteasome pathway, small RNA pathways, cell division cycle components, and the epigenetic mechanism as defense responses during plant–pathogen interactions. Capturing the information on these pathways for the development of strategies against geminivirus infection is argued to provide the basis for new genetic approaches to resistance.Item Recent advances in plant-virus interaction with emphasis on small interfering RNAs (siRNAs)(Springer, 2013) Sharma, Namisha; Sahu, Pranav Pankaj; Puranik, Swati; Prasad, ManojRegulation of several biological functions in plants has now been known to involve diverse RNA silencing pathways. These vital pathways involve various components such as dsRNA, Dicer, RNA-dependent RNA polymerase and Argonaute proteins, which lead to the production of several small RNAs (sRNAs) varying in their sizes. These sRNAs have significant role in the regulation of gene expression at transcriptional and translational levels. Among them, small interfering RNAs (siRNAs; majorly 21, 22 and 24 nt) have been shown to play an important role in plants' resistance against many viruses by inhibiting the viral gene expression. Furthermore, it has also been highlighted that siRNA-mediated methylation of viral DNA confers resistance to various plant DNA viruses. In this review, we have outlined the recent advances made using the siRNA-mediated antiviral strategy, along with methylation-based epigenetic defensive mechanisms as a protective measure against diverse plant viruses.Item Epigenetic mechanisms of plant stress responses and adaptation(Springer, 2013) Sahu, Pranav Pankaj; Pandey, Garima; Sharma, Namisha; Puranik, Swati; Muthamilarasan, Mehanathan; Prasad, ManojEpigenetics has become one of the hottest topics of research in plant functional genomics since it appears promising in deciphering and imparting stress-adaptive potential in crops and other plant species. Recently, numerous studies have provided new insights into the epigenetic control of stress adaptation. Epigenetic control of stress-induced phenotypic response of plants involves gene regulation. Growing evidence suggest that methylation of DNA in response to stress leads to the variation in phenotype. Transposon mobility, siRNA-mediated methylation and host methyltransferase activation have been implicated in this process. This review presents the current status of epigenetics of plant stress responses with a view to use this knowledge towards engineering plants for stress tolerance.Item Comprehensive genome-wide survey, genomic constitution and expression profiling of the NAC transcription factor family in foxtail millet (Setaria italica L.)(PLOS, 2013) Puranik, Swati; Sahu, Pranav Pankaj; Mandal, Sambhu Nath; Suresh B., Venkata; Parida, Swarup K.; Prasad, ManojThe NAC proteins represent a major plant-specific transcription factor family that has established enormously diverse roles in various plant processes. Aided by the availability of complete genomes, several members of this family have been identified in Arabidopsis, rice, soybean and poplar. However, no comprehensive investigation has been presented for the recently sequenced, naturally stress tolerant crop, Setaria italica (foxtail millet) that is famed as a model crop for bioenergy research. In this study, we identified 147 putative NAC domain-encoding genes from foxtail millet by systematic sequence analysis and physically mapped them onto nine chromosomes. Genomic organization suggested that inter-chromosomal duplications may have been responsible for expansion of this gene family in foxtail millet. Phylogenetically, they were arranged into 11 distinct sub-families (I-XI), with duplicated genes fitting into one cluster and possessing conserved motif compositions. Comparative mapping with other grass species revealed some orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of genes. The evolutionary significance as duplication and divergence of NAC genes based on their amino acid substitution rates was understood. Expression profiling against various stresses and phytohormones provides novel insights into specific and/or overlapping expression patterns of SiNAC genes, which may be responsible for functional divergence among individual members in this crop. Further, we performed structure modeling and molecular simulation of a stress-responsive protein, SiNAC128, proffering an initial framework for understanding its molecular function. Taken together, this genome-wide identification and expression profiling unlocks new avenues for systematic functional analysis of novel NAC gene family candidates which may be applied for improvising stress adaption in plants.Item Transcription factors: modulating plant adaption in the scenario of changing climate(Springer, 2013) Puranik, Swati; Prasad, ManojClimate change and the associated environmental constraints have created a challenge to sustain food security and biodiversity globally. The response to adverse environments is a complex process and plants integrate several approaches that allow them to withstand climatic restraints, depending on the timing and length. Changes at the transcriptional level of various genes transduce cellular signals for the synthesis of necessary metabolites. Transcription factors have the principal role for the efficient adaptation capacity of the plants, and present an attractive target category for manipulation and gene regulation. As many biological processes in plants are regulated at the level of transcription, understanding transcription factor function is an important step towards understanding plant responses to environmental conditions. Among them, numerous transcription factors belonging to several large transcription factor families, such as AP2/ERF, bZIP, MYB, MYC, Cys2His2 zinc finger, WRKY, and NAC, have been shown as stress-responsive proteins. They act both by an abscisic acid-dependent and -independent manner, and through their interaction with respective cis-elements of their target genes, play crucial roles in recuperating plant stress tolerance. We highlight the considerable biotechnological progress made towards understanding the molecular stress responses of plants using important transcription factor families. The progress of their practical and application value in crop improvement through genetic engineering is also discussed.Item Transcript profiling identifies novel transcripts with unknown functions as primary response components to osmotic stress in wheat (Triticum aestivum L.)(Springer, 2013) Garg, Bharti; Puranik, Swati; Misra, Shrilekha; Tripathi, Bhumi Nath; Prasad, ManojOsmotic stress induced by dehydration and salinity, is among the major abiotic stresses that adversely impacts crop productivity and plants often display cultivar-dependent response against osmotic imbalance. To better understand the molecular mechanisms underlying differential responses to dehydration, transcriptome changes of two contrasting wheat (Triticum aestivum L.) cultivars were evaluated in plants grown under unfavorable osmotic conditions. A total of 107 non-redundant transcripts were identified. Of these, most had unknown functions (31; ~30 %) signifying the existence of putative stress-specific genes in wheat, reported here for the first time. Upon comparing with previous transcriptomic studies, 43 (40 %) of the osmotically-responsive transcripts were found not to be documented. These new transcripts may therefore signify unexplored gene sources for specific responses towards short-term osmotic stress in wheat. Through macroarray analysis, 69 (~64 %) transcripts were found to be differentially expressed (≥3-fold) and expression of 14 transcripts (with known or unknown functions) was further confirmed by quantitative real time PCR. Expression analysis of the seven unknown transcripts also revealed their tissue- and stress-specific regulation. Comparative in silico mapping of these 107 wheat transcripts against available mapping data for rice (40; ~37 %), maize (34; ~32 %), and sorghum (33; ~31 %) revealed presence of wheat orthologous sequences in these cereal crops. This study provides an interesting account on several novel genes, besides those with known functions, which may regulate stress response dynamics and thus, may be used as potential candidates to improve stress adaptability through genetic and molecular studies.Item Dynamics of defense related components in two contrasting genotypes to tomato upon infection with tomato leaf curl New Delhi virus(Springer, 2012) Sahu, Pranav Pankaj; Rai, Neeraj Kumar; Puranik, Swati; Roy, Anirban; Khan, Moinuddin; Prasad, ManojTomato leaf curl virus (ToLCV) disease is a serious threat for tomato cultivation in the tropics and subtropics. Despite serious efforts no immune commercial varieties or F(1) hybrids are available till date. In this study, the interaction between Solanum lycopersicum and ToLCV was characterized on molecular and biochemical basis. RNA silencing mediated by short interfering RNA (siRNA) and reactive oxygen species (ROS) has been proposed as central components of plant adaptation to several stresses. A comparative RNA interference study between two contrasting tomato genotypes, LA1777 (tolerant) and 15SBSB (susceptible) infected with Tomato Leaf Curl New Delhi Virus (ToLCNDV) revealed relatively higher accumulation of siRNA in the leaves of tolerant genotype. In LA1777, ToLCNDV produced chlorotic as well as necrotic areas at the inoculation sites 5-10 days post-inoculation. Caspase-9- and caspase-3-like activities were significantly increased in response to ToLCNDV infection in LA1777 at inoculated region. Activities of antioxidant enzymes involved in the detoxification of ROS were examined in both systemic and localized area of infection, and their expression level was further validated through quantitative real-time PCR of the corresponding transcripts. Expression patterns of three genes encoding pathogenesis-related proteins showed higher accumulation in tolerant genotype. Tolerance against the ToLCNDV in LA1777 can be attributed to the higher siRNA accumulation, localized cell death, altered levels of antioxidant enzymes and activation of pathogenesis-related genes at different durations of virus infection. Based on these direct and indirect evidences, we have proposed a putative mechanism for ToLCNDV tolerance in the tolerant genotype.Item Recent advances in tomato functional genomics: utilization of VIGS(Springer, 2012) Sahu, Pranav Pankaj; Puranik, Swati; Khan, Moinuddin; Prasad, ManojTomato unquestionably occupies a significant position in world vegetable production owing to its world-wide consumption. The tomato genome sequencing efforts being recently concluded, it becomes more imperative to recognize important functional genes from this treasure of generated information for improving tomato yield. While much progress has been made in conventional tomato breeding, post-transcriptional gene silencing (PTGS) offers an alternative approach for advancement of tomato functional genomics. In particular, virus-induced gene silencing (VIGS) is increasingly being used as rapid, reliable, and lucrative screening strategy to elucidate gene function. In this review, we focus on the recent advancement made through exploiting the potential of this technique for manipulating different agronomically important traits in tomato by discussing several case studies.