VIGS and SIGS-mediated PDS gene silencing in dicot weeds reveals species-specific responses and delivery limitations.

RNA interference (RNAi) is a promising approach for pest control but remains underexplored in weed management. The current study evaluated two transient RNAi approaches, virus induced gene silencing (VIGS) in the noxious weed species Palmer amaranth, waterhemp, and black nightshade, and spray induced gene silencing (SIGS) in Palmer amaranth, to silence the Phytoene desaturase (PDS) gene, as a visual reporter. For VIGS, species-specific PDS fragments were cloned into the Tobacco rattle virus (TRV) vector and delivered either directly via Agrobacterium-mediated agroinfiltration or indirectly through sap inoculation using infected Nicotiana benthamiana. In Palmer amaranth, indirect VIGS resulted in a tenfold reduction in PDS gene expression and induced visible silencing phenotypes, including bleaching, chlorosis, and stunting, whereas direct application did not produce detectable symptoms. In contrast, no discernible responses were observed for VIGS in waterhemp and black nightshade. SIGS using dsPaPDS at 2 μM and 3 μM concentrations induced transient bleaching in newly emerging leaves and sustained growth reduction, achieving up to 83% phenotypic response; however, no significant reduction in PDS gene expression was detected by qRT-PCR. Overall, under the conditions evaluated in this study, indirect VIGS was the most effective approach for PDS gene silencing in Palmer amaranth. These findings highlight species-specific responses and delivery constraints of RNAi-based approaches and emphasize the need for improved dsRNA delivery systems to advance RNAi-mediated weed management.