Agathisflavone as a novel natural inhibitor of ToLCNDV suppressor proteins: insights from computational analyses and in planta validation.

Tomato leaf curl New Delhi virus (ToLCNDV) is a major begomovirus in tomato, where suppressor proteins AV2, AC2, and AC4 counteract host RNA silencing and intensify pathogenicity. Current management largely depends on vector control and resistance breeding, both limited by environmental concerns and rapid viral evolution, underscoring the need for targeted antiviral agents. Fifteen plant-derived phytochemicals, previously reported to exhibit antiviral activity against human viruses and representing major phytochemical classes such as flavonoids, terpenoids, alkaloids, and phenolics, were screened against the suppressor proteins AV2, AC2, and AC4 encoded by the DNA-A genomic component of ToLCNDV. Based on molecular docking scores, the top-performing candidates comprised six compounds spanning these classes. Among them, agathisflavone consistently displayed the strongest binding affinities, with binding energies of - 8.7, - 9.0, and - 7.9 kcal/mol for AV2, AC2, and AC4, respectively, highlighting it as the most promising inhibitor. Molecular dynamics simulations showed stable protein-ligand interactions with low RMSD and RMSF values and favourable van der Waals and electrostatic contributions. An agathisflavone-enriched extract from cashew leaves was formulated for spray application. In planta silencing reversal assays conducted under agathisflavone-enriched formulation-treated conditions demonstrated strong inhibition of AV2 and AC2 suppressor functions and moderate inhibition of AC4, thereby restoring host RNA silencing. These findings establish agathisflavone as the most potent natural antiviral among the tested phytochemicals and provide a rational framework for developing sustainable antiviral strategies against begomoviruses.