A novel pepper mild mottle virus-based vector for high-efficiency gene expression and silencing in pepper (Capsicum annuum)

Abstract

Abstract Background Pepper ( Capsicum annuum ) is an economically important vegetable crop worldwide. However, progress in its functional genomics has been severely impeded by the low efficiency and time-consuming nature of stable genetic transformation. To address these limitations, plant virus-based vectors have been developed as potent alternatives. This study aimed to construct a novel, highly efficient viral vector system based on the pepper mild mottle virus (PMMoV), a pathogen that induces only mild symptoms and is naturally adapted to pepper, for both gene expression and virus-induced gene silencing (VIGS). Results We successfully developed a novel PMMoV-based vector, designated pKL736. For exogenous gene expression, pKL736-GFP produced intense fluorescence in systemically infected leaves across various pepper cultivars. Regarding VIGS, we utilized the phytoene desaturase ( PDS ) gene as a visual marker and determined that a ~ 150 bp insert (pKL736-PDS156) achieved optimal silencing efficiency. This construct induced pronounced photobleaching in leaves of two pepper cultivars, Zunla-1 and JS, at 20 days post-inoculation. Notably, pKL736-PDS156 also mediated robust silencing in pepper fruits, with silencing efficiencies reaching 90.7% and 85.5% in Zunla-1 and JS, respectively. The PMMoV-based VIGS system demonstrated robust versatility, extending to five additional pepper varieties with consistent silencing efficiencies exceeding 80% in both leaf and fruit tissues. Furthermore, application of this vector to silence the eukaryotic translation initiation factor 4E ( eIF4E ) gene resulted in significantly enhanced resistance against two potyviruses. Conclusions The PMMoV-based vector represents a significant methodological advancement for rapid, high-throughput functional genomics in pepper. Its ability to deliver consistent and potent gene silencing in both vegetative and reproductive tissues across diverse cultivars, establishes it as a powerful tool for accelerating gene function validation in pepper, especially for screening disease-resistant genes.