Research Projects

Okra (Abelmoschus esculentus (L.) Moench), a key vegetable crop in India, contributes over 72.9% to global production. However, its productivity is severely impacted by viral diseases, particularly Okra enation leaf curl disease (OELCuD), caused by Okra enation leaf curl virus (OELCuV), a monopartite begomovirus transmitted by the Bemisia tabaci. OELCuV has been reported to cause yield losses ranging from 5% to 74% in India. Despite its significance, the molecular mechanisms that allow OELCuV to overcome host defenses remain poorly understood (Kumar et al. 2024; Kumar et al. 2025). RNA silencing is a vital antiviral defense mechanism in plants, wherein small interfering RNAs (siRNAs) target viral RNA for degradation. To counteract this, begomoviruses encode RNA silencing suppressor (RSS) proteins (Kumar et al. 2015). In related viruses such as MYMIV and ToLCV, proteins AC2, AC4, and AV2 have been experimentally validated as RSSs. Genome annotations suggest that OELCuV also encodes these proteins, but their roles have not been functionally characterized (Sundaresan et al. 2020). This project aims to identify and functionally characterize the RSS activity of OELCuV-encoded AC2, AC4, and AV2 proteins using an integrated molecular and computational approach. The study will begin with in silico analysis of their sequences to predict structural features and subcellular localization using tools like Modeller, TMHMM, and cNLS Mapper. The ORFs will be PCR-amplified and cloned into the pBI121 binary vector under the CaMV 35S promoter, followed by Agrobacterium tumefaciens transformation for agroinfiltration studies (Sundaresan et al. 2020). Functional validation will be performed using GFP-silenced Nicotiana tabacum plants. The ability of the OELCuV proteins to reverse GFP silencing will be monitored via UV fluorescence, and suppressor strength quantified by RT-qPCR and siRNA detection. Localization of viral proteins will be confirmed using confocal microscopy with fluorescent tags (GFP/RFP). Protein expression will be validated by SDS-PAGE and western blotting (Gong et al. 2021; Zhao et al. 2024). Additionally, a translational objective involves standardizing non-transgenic delivery systems using spray-induced gene silencing (SIGS) and green-synthesized nanoparticles. dsRNA constructs targeting viral genes will be delivered into Arabidopsis and okra tissues to evaluate uptake and antiviral effects (Chang et al. 2013; Sarkar and Barman 2021; Singh et al. 2024). The outcomes will provide the first functional and structural insights into OELCuV’s silencing suppressors. This knowledge will be instrumental in designing RNAi-based resistance strategies, such as transgenic okra lines or SIGS platforms, and may support breeding programs by identifying molecular markers linked to resistance traits. The project addresses a critical gap in begomovirus research and aligns with national priorities in plant health, food security, and sustainable agriculture.