Publications of NIPGR Scientists

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Author: search.filters.author.Prasad, ManojSubject: search.filters.subject.Tomato leaf curl New Delhi virus

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    The sly-miR166-SlyHB module acts as a susceptibility factor during ToLCNDV infection
    (Springer Nature Publishing AG, 2022) Prasad, Ashish; Sharma, Namisha; Chirom, Oceania; Prasad, Manoj
    Tomato leaf curl New Delhi virus (ToLCNDV) is a devastating pathogen that causes huge crop loss. It is spreading to new geographical locations at a very rapid rate-raising serious concerns. Evolution of insecticidal resistance in Bemisia tabaci which acts as the carrier for ToLCNDV has made insect control very difficult in the recent years. Thus, it is important that the host molecular mechanisms associated with ToLCNDV resistance/susceptibility are investigated to develop management strategies. In our study, we have identified that sly-miR166/SlyHB module acts as a susceptibility factor to ToLCNDV in Solanum lycopersicum. Sly-miR166 is differentially regulated upon ToLCNDV infection in two contrasting tomato cultivars; H-88-78-1 (tolerant to ToLCNDV) and Punjab Chhuhara (susceptible to ToLCNDV). Expression analysis of predicted sly-miR166 targets revealed that the expression of SlyHB is negatively correlated with its corresponding miRNA. Virus-induced gene silencing of SlyHB in the susceptible tomato cultivar resulted in the decrease in disease severity suggesting that SlyHB is a negative regulator of plant defence. In summary, our study highlights a miRNA/target module that acts as a susceptibility factor during ToLCNDV infection. To the best of our knowledge, this is the first report that highlights the role of sly-miR166/SlyHB module in ToLCNDV pathogenesis.
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    Molecular characterization of SlATG18f in response to Tomato leaf curl New Delhi virus infection in tomato and development of a CAPS marker for leaf curl disease tolerance
    (Springer Nature Publishing AG, 2021) Prasad, Ashish; Hari‑Gowthem, Gunaseelen; Muthamilarasan, Mehanathan; Hussain, Zakir; Yadav, Pawan Kumar; Tripathi, Sandhya; Prasad, Manoj
    Autophagy is a highly conserved catabolic process regulating cellular homeostasis and adaptation to different biotic and abiotic stress. Several autophagy-related proteins (ATGs) are reported to be involved in autophagic processes, and considering their importance in regulating growth and stress adaptation, these proteins have been identified and characterized in several plant species. However, there is no information available on the role of autophagy-related proteins regulating the tolerance of tomato to tomato leaf curl disease (ToLCD). Given this, the present genome-wide study identified thirty ATG-encoding genes (SlATG) in tomato, followed by their functional characterization. Expression profiling of the SlATG genes in contrasting tomato cultivars subjected to virus infection showed a 4.5-fold upregulation of SlATG18f in the tolerant cultivar. Further, virus-induced gene silencing of SlATG18f in the tolerant cultivar conferred disease susceptibility, which suggested the role of this gene in Tomato leaf curl New Delhi virus tolerance. Comparison of the gene sequence of both tolerant and susceptible cultivars along with the 5′ upstream regions identified an SNP (A/T) at -2916 upstream of the start codon. A cleaved amplified polymorphic sequence (CAPS) marker was developed targeting this region, which showed a significant association with the tolerance characteristics in the tomato germplasm (R2 = 0.1787). Altogether, the study identified a potential gene that could be used to develop ToLCNDV tolerant tomato cultivars using transgene-based or marker-assisted breeding-based approaches.
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    Genomic dissection of ROS detoxifying enzyme encoding genes for their role in antioxidative defense mechanism against Tomato leaf curl New Delhi virus infection in tomato
    (Elsevier B.V., 2021) Sharma, Namisha; Muthamilarasan, Mehanathan; Dulani, Priya; Prasad, Manoj
    In the present study, genes encoding for six major classes of enzymatic antioxidants, namely superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), Peroxidase (Prx) and glutathione S-transferase (GST) are identified in tomato. Their expression was studied in tomato cultivars contrastingly tolerant to ToLCNDV during virus infection and different hormone treatments. Significant upregulation of SlGR3, SlPrx25, SlPrx75, SlPrx95, SlGST44, and SlGST96 was observed in the tolerant cultivar during disease infection. Virus-induced gene silencing of SlGR3 in the tolerant cultivar conferred disease susceptibility to the knock-out line, and higher accumulation (~80%) of viral DNA was observed in the tolerant cultivar. Further, subcellular localization of SlGR3 showed its presence in cytoplasm, and its enzymatic activity was found to be increased (~65%) during ToLCNDV infection in the tolerant cultivar. Knock-out lines showed ~3- and 3.5-fold reduction in GR activity, which altogether underlines that SlGR3 is vital component of the defense mechanism against ToLCNDV infection.
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    Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato
    (Springer Nature Publishing AG, 2020) Sharma, Namisha; Prasad, Manoj
    Key message: Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato. Abstract: Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼ 40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.
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    Characterization of DEAD-box family of RNA helicases in tomato provides insights into their roles in biotic and abiotic stresses
    (Elsevier B.V., 2019) Pandey, Saurabh; Muthamilarasan, Mehanathan; Sharma, Namisha; Chaudhry, Vaishali; Dulani, Priya; Shweta, Shweta; Jha, Sarita; Mathur, Saloni; Prasad, Manoj
    In plants, RNA helicases play significant roles in growth, development and stress response. In a previous study, a three-fold upregulation of a DEAD-box RNA helicase in a tomato cultivar tolerant to Tomato leaf curl New Delhi virus (ToLCNDV) as compared to susceptible cultivar during virus infection was shown. Given this, a comprehensive study was performed to identify the members of RNA helicase family in tomato and analyze their functional properties in response to abiotic stresses, hormone treatments and ToLCNDV infection. A total of 131 genes were identified and classified into DEAD- (42), DEAH- (38), and DExD/H-box (51) RNA helicases. Expression profiling of candidate genes in response to abiotic stresses and ToLCNDV infection in contrasting tomato cultivars suggested the putative roles of SlDEAD23 and SlDEAD35 in biotic and abiotic stresses. Heterologous overexpression of these genes in yeast enhanced the tolerance of transgenic cells to salt and cold stresses. Further, virus-induced silencing of SlDEAD35 in ToLCNDV tolerant cultivar resulted in susceptibility to virus infection, thus suggesting its involvement in tolerance mechanism. Altogether, this study provides novel insights into the structure, organization and involvement of DEAD-box RNA helicase genes in biotic and abiotic stress responses in tomato.
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    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, Manoj
    Tomato 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.