• dsRNA
• Lactuca sativa
• PTGS
• RNA interference

RNA interference is a promising biotechnological tool that involves the external application of dsRNAs to plants and is emerging as a new strategy to silence endogenous plant genes and control plant disease. However, the effects of externally applied dsRNAs on endogenous gene regulation in plants are still poorly studied. This study investigated the application of dsRNAs to silence endogenous genes in lettuce (Lactuca sativa). We used Escherichia coli HT115 (DE3) and the vector L4440 to produce dsRNAs targeting LsGUN4 and LsChlH. Two application methods were tested: seeds or seedling dip and leaf infiltration. While no phenotypic changes were observed during seed or seedling soaking, more or less evident chlorosis appeared in plants infiltrated with LsGUN4 and LsChlH. The effects of gene silencing were assessed at 48 and 96 h post-treatment (hpt) by measuring chlorophyll and carotenoid content and analyzing the relative expression levels of target genes. No statistical difference in photosynthetic pigment content was observed between treated and control plants. In contrast, LsGUN4 mRNA levels showed a significant reduction at both 48 and 96 hpt compared to GFP-dsRNA treated control plants, while LsChlH mRNA levels showed a significant reduction only at 96 hpt. Our results provide valuable insights into the efficacy of dsRNA-mediated gene silencing in non-model plants and help to optimize the conditions for applying endogenous gene silencing in economically important crops.